While attending the University of Tennessee, Mayo played for the Tennessee Volunteers football team from 2004 to 2007. After redshirting the 2004 season, he appeared in six games at weak-side outside linebacker in 2005, finishing with 13 tackles (10 solo). Mayo made the Volunteers starting lineup as a redshirt sophomore in 2006, as he started 11 contests at weak-side outside linebacker. He finished third on the team with 83 tackles (48 solos), including five sacks for minus 40 yards, 12.5 stops for losses of 51 yards and a quarterback pressure. He also recovered one fumble and deflected a pass. Rivals.com subsequently named him to their All-American second team.

For his junior season, Mayo moved to middle linebacker and started all 14 games. Serving as the defensive squad’s co-captain, he went on to register 140 tackles in 2007, the most by a Tennessee defender since Earnest Fields registered those same totals for the Volunteers in 1990. He added 1.5 quarterback sacks for minus 11 yards, 8.5 stops for losses and five quarterback pressures, and also returned an interception 34 yards for a touchdown. Mayo was a first-team All-Southeastern Conference selection and also earned All-American second-team honors by The NFL Draft Report.

Mayo was considered one of the best linebackers available in the 2008 NFL Draft and drew comparisons to Will Witherspoon. Sporting News described Mayo as a “perfect fit to play one of the inside spots” in a 3-4 defense.

The New England Patriots selected Mayo in the first round (10th overall) of the 2008 NFL Draft. Mayo was the second linebacker drafted in 2008, behind Keith Rivers.

On July 24, 2008, the New England Patriots signed Mayo to a five-year contract (rather than the six-year maximum allowed by the NFL’s collective bargaining agreement) worth $18.9 million, including $13.8 million in bonuses and guarantees. Mayo was the only rookie in the Patriots’ 2008 class to start in Week 1, and played every snap for the defense.

Mayo was named the NFL’s Defensive Rookie of the Month for October 2008. He led the Patriots with 24 tackles for the month, including 11 against the Denver Broncos in his first Monday Night Football appearance. In the Patriots’ Thursday Night Football game on November 13, 2008, against their division rivals, the New York Jets, Mayo led all defensive players with 20 tackles (16 solo, 4 assisted), the first 20-tackle game of his career.

Mayo has two brothers, both of whom played college football as linebackers. His younger brother, Deron Mayo, played in the Canadian Football League, and is currently the assistant strength and conditioning coach for the New England Patriots. His brother Derek Mayo is a graduate of Richmond, where he won the NCAA Football Championship Subdivision title in 2008.

In 2010, Mayo was named a defensive captain. In Week 4 against the Miami Dolphins, Mayo recorded 16 tackles, and two weeks later notched 18 tackles in an overtime win over the Baltimore Ravens. In Week 8, Mayo recorded 14 tackles in a win over the Minnesota Vikings. Against the Indianapolis Colts in Week 11, Mayo recorded 15 tackles in a win. In Week 12, Mayo’s eight tackles gave him 132 on the season, surpassing his previous career high of 128, set in 2008. He added another 12 tackles in Week 13 against the New York Jets, and had 16 in Week 15 against the Green Bay Packers. He ended the season with a league-high 175 tackles, along with two sacks and one forced fumble.

He was named as a reserve to the 2011 Pro Bowl on January 2, 2011. In the same season he was named to the 2010 All Pro team.

On December 17, 2011, at the beginning of Week 15, it was announced that he had signed a 5-year contract extension with the Patriots.

In 2011, he missed 3 games due to injuries but still managed to eclipse the 100-tackle mark. Mayo and the Patriots reached Super Bowl XLVI. In the game, Mayo had 11 tackles but the Patriots lost to the New York Giants by a score of 21–17.

On November 16, 2012, Mayo was fined $10,000 for a late hit out of bounds on Week 10 against C. J. Spiller with the Buffalo Bills. This drew an unnecessary roughness call.

Later in the year, he was selected in the 2013 Pro Bowl in recognition of his successful 2012 season.

Mayo’s playing time dipped in the 2015 season, being behind Dont’a Hightower and Jamie Collins on the depth chart. On January 19, 2016, days before the AFC Championship against the Denver Broncos, Mayo was placed on injured reserve with a shoulder injury. 2015 was the third consecutive year his season ended early.

On February 16, 2016, Mayo posted a message on his Instagram account announcing his retirement, thanking the Patriots for the previous eight years.

On March 27, 2019, Mayo was hired by the New England Patriots to be their inside linebackers coach.