Hefner was born in Chicago on April 9, 1926, the first child of Glenn Lucius Hefner (1896–1976), an accountant, and his wife Grace Caroline (Swanson) Hefner (1895–1997) who worked as a teacher. His parents were from Nebraska. He had a younger brother, Keith (1929–2016). His mother was of Swedish ancestry, and his father was German and English.

He attended Sayre Elementary School and Steinmetz High School, then served from 1944 to 1946 as a U.S. Army writer for a military newspaper. Hefner graduated from the University of Illinois at Urbana–Champaign in 1949 with a Bachelor of Arts in Psychology and a double minor in Creative Writing and Art, having earned his degree in two and a half years. After graduation, he took a semester of graduate courses in Sociology at Northwestern University, but dropped out soon after.

Hefner was known to friends and family simply as “Hef”. He married Northwestern University student Mildred (“Millie”) Williams in 1949, and they had daughter Christie (b. 1952) and son David (b. 1955). Before the wedding, Mildred confessed that she had an affair while he was away in the army. He called the admission “the most devastating moment of my life.” A 2006 E! True Hollywood Story profile of Hefner revealed that Mildred allowed him to have sex with other women, out of guilt for her own infidelity and in the hope that it would preserve their marriage. The two were divorced in 1959.

In January 1952, Hefner left his job as a copywriter for Esquire after he was denied a $5 raise. In 1953, he took out a mortgage loan of $600 and raised $8,000 from 45 investors (including $1,000 from his mother—”not because she believed in the venture,” he told E! in 2006, “but because she believed in her son”) to launch Playboy, which was initially going to be called Stag Party. The first issue was published in December 1953 and featured Marilyn Monroe from a 1949 nude calendar shoot she did under a pseudonym when she was in financial distress before she was famous. Monroe did not consent to her pictures to be used by Hefner and she was not compensated by Hefner for them. That first issue sold more than 50,000 copies. (Hefner never met Monroe, but he bought the crypt next to hers at the Westwood Village Memorial Park Cemetery in 1992 for $75,000.)

Esquire magazine rejected Charles Beaumont’s science fiction story “The Crooked Man” in 1955, so Hefner agreed to publish it in Playboy. The story highlighted straight men being persecuted in a world where homosexuality was the norm. The magazine received angry letters, so Hefner responded, “If it was wrong to persecute heterosexuals in a homosexual society then the reverse was wrong, too.” In 1961, Hefner watched Dick Gregory perform at the Herman Roberts Show Bar in Chicago, and he hired Gregory to work at the Chicago Playboy Club. Gregory attributed the launch of his career to that night.

On June 4, 1963, Hefner was arrested for promoting obscene literature after he published an issue of Playboy featuring nude shots of Jayne Mansfield in bed with a man present. The case went to trial and resulted in a hung jury.

In the 1960s, Hefner created “private key” clubs that were racially diverse. During the civil rights movement in 1966, Hefner sent Alex Haley to interview George Lincoln Rockwell, much to Rockwell’s surprise because Haley was black. Rockwell had founded the American Nazi Party, and was described as the “American Hitler” by the BBC. Rockwell agreed to meet with Haley only after gaining assurance that he was not Jewish, although Rockwell kept a handgun on the table throughout the interview. The interview was recreated in Roots: The Next Generations in 1979, with James Earl Jones as Haley and Marlon Brando as Rockwell; Brando won a Primetime Emmy Award for his portrayal of Rockwell. Haley had also interviewed Malcolm X in 1963 and Martin Luther King Jr. in 1966 for the newly established 1962 “playboy interview”; all three interviewees were assassinated by 1968.

Hefner debated The Playboy Philosophy with William F. Buckley Jr. on Firing Line in Episode 026, Recorded on September 12, 1966.

In 1970, Hefner stated that “militant feminists” are “unalterably opposed to the romantic boy-girl society that Playboy promotes” and ordered an article in his magazine against them. In the 1993 The Simpsons episode “Krusty Gets Kancelled”, Hefner voiced himself. In 1999, Hefner financed the Clara Bow documentary Discovering the It Girl. “Nobody has what Clara had,” he said. “She defined an era and made her mark on the nation”. Hefner guest-starred as himself in the 2000 Sex and the City episode “Sex and Another City”. In 2005, he guest-starred on the HBO shows Curb Your Enthusiasm and Entourage. He guest-starred as himself in a 2006 episode of Seth Green’s Robot Chicken on the late-night programming block Adult Swim. In the 2007 Family Guy episode “Airport ’07”, he voiced himself. He has a star on the Hollywood Walk of Fame for television and made several movie appearances as himself. In 2009, he received a “worst supporting actor” nomination for a Razzie award for his performance as himself in Miss March. On his official Twitter account, he joked about this nomination: “Maybe I didn’t understand the character.”

Hefner remade himself as a bon vivant and man about town, a lifestyle that he promoted in his magazine and TV shows. He admitted to being “‘involved’ with maybe eleven out of twelve months’ worth of Playmates” during some years. Donna Michelle, Marilyn Cole, Lillian Müller, Shannon Tweed, Barbi Benton, Karen Christy, Sondra Theodore, and Carrie Leigh were a few of his many lovers; Leigh filed a $35 million palimony suit against him. In 1971, he acknowledged that he experimented in bisexuality. Also in 1971, he established a second residence in Los Angeles with the acquisition of Playboy Mansion West, and he moved there permanently from Chicago in 1975.

In 1978, Hefner helped organize fund-raising efforts that led to the restoration of the Hollywood Sign. He hosted a gala fundraiser at the Playboy Mansion and contributed $27,000 (or 1/9 of the total restoration costs) by purchasing the letter Y in a ceremonial auction.

On March 7, 1985, Hefner had a minor stroke at age 58, and he re-evaluated his lifestyle and made several changes. He toned down the wild, all-night parties, and daughter Christie took over the operation of the Playboy empire in 1988. The following year, he married Playmate of the Year Kimberley Conrad; they were 36 years apart in age. The couple had sons Marston Glenn (born 1990) and Cooper (born 1991). The E! True Hollywood Story profile noted that the notorious Playboy Mansion had been transformed into a family-friendly homestead. He and Conrad separated in 1998 and she moved into the house next door to the mansion. Hefner filed for divorce from Conrad in 2009 after an 11-year separation, citing irreconcilable differences. He has stated that he only remained nominally married to her for the sake of their children, and their youngest child had just turned 18. The divorce was finalized in 2010.

He is interred at Westwood Memorial Park in Los Angeles, in the crypt beside Marilyn Monroe, for which he paid $75,000 in 1992. “Spending eternity next to Marilyn is an opportunity too sweet to pass up,” Hefner had told the Los Angeles Times in 2009.

Hefner donated $100,000 to the University of Southern California’s School of Cinematic Arts to create a course called “Censorship in Cinema”, and $2 million to endow a chair for the study of American film. In 2007, the university’s audiovisual archive at the Norris Theater received a donation from Hefner and was renamed to the Hugh M. Hefner Moving Image Archive in his honor.

Hefner became known for moving an ever-changing coterie of young women into the Playboy Mansion, including twins Mandy and Sandy Bentley. He dated as many as seven women concurrently. He also dated Brande Roderick, Izabella St. James, Tina Marie Jordan, Holly Madison, Bridget Marquardt, and Kendra Wilkinson. Madison, Wilkinson, and Marquardt appeared on The Girls Next Door depicting their lives at the Playboy Mansion. In October 2008, all three of them decided to leave the mansion.

In January 2009, Hefner began a relationship with Crystal Harris; she joined the Shannon Twins after his previous “number one girlfriend” Holly Madison had ended their seven-year relationship. On December 24, 2010, he became engaged to Harris, but she broke off their engagement on June 14, 2011, five days before their planned wedding. The July issue of Playboy reached store shelves and customer’s homes within days of the wedding date; it featured Harris on the cover and in a photo spread, as well. The headline on the cover read “Introducing America’s Princess, Mrs. Crystal Hefner”. Hefner and Harris subsequently reconciled and married on December 31, 2012.

Brigitte Berman’s documentary Hugh Hefner: Playboy, Activist and Rebel was released on July 30, 2010. He had previously granted full access to documentary filmmaker and television producer Kevin Burns for the A&E Biography special Hugh Hefner: American Playboy in 1996. Hefner and Burns later collaborated on numerous other television projects, most notably on The Girls Next Door, a reality series that ran for six seasons (2005–2009) and 90 episodes. Hefner also made a voice-only appearance as himself in the 2011 film Hop.

On November 18, 2010, Children of the Night founder and president Dr. Lois Lee presented Hefner with the organization’s first-ever Founder’s Hero of the Heart Award in appreciation for his unwavering dedication, commitment and generosity.

On April 26, 2010, Hefner donated the last $900,000 sought by a conservation group for a land purchase needed to stop the development of the famed vista of the Hollywood Sign. Sylvilagus palustris hefneri, an endangered subspecies of marsh rabbit, is named after him in honor of financial support that he provided.

He donated and raised money for the Democratic Party. In 2011, he referred to himself as an independent due to dissatisfaction with both the Democratic and Republican parties. Nonetheless, in 2012, he supported Barack Obama’s reelection campaign.

In 2012, Hefner announced that his youngest son Cooper would succeed him as the public face of Playboy.

Hefner’s brother Keith died at age 87 on April 8, 2016, one day before Hefner’s 90th birthday.

In January 2016, the Playboy Mansion was put on the market for $200 million, on condition that Hugh Hefner would continue to work and live in the mansion. Later that year it was sold to Daren Metropoulos, a principal at private equity firm Metropoulos & Company, for $100 million. Metropoulos planned to reconnect the Playboy Mansion property with a neighboring estate that he purchased in 2009, combining the two for a 7.3 acre (3-hectare) compound as his own private residence.

In May 2017, Eugena Washington was the last Playmate of the Year to be announced by Hugh Hefner at the Playboy Mansion.

Hefner died at his home in Holmby Hills, Los Angeles, California, on September 27, 2017, at the age of 91. The cause was sepsis brought on by an E. coli infection.

On October 3, 2017, Playboy Enterprises announced that a Hugh Hefner biopic directed by Brett Ratner with the screenplay by Jeff Nathanson was greenlit with Jared Leto rumored to play Hefner. It was indefinitely put on hold following sexual harassment allegations against Ratner on November 2, 2017 and Leto’s representatives stated that reports of him being attached to the film at any point were false.

The Amazon original series American Playboy: The Hugh Hefner Story was released in 2017. It stars Matt Whelan in the title role, along with Emmett Skilton and Chelsie Preston Crayford. The first season was released on April 7, composed of ten episodes. The series is a combination of interviews, archival footage (including moments found in Hefner’s vast personal collection), and cinematic re-enactments that cover the launch of the magazine as well as the next six decades of Hefner’s personal life and career. The series was filmed in Auckland, New Zealand.