Casilla was originally signed by the Oakland A’s on January 31, 2000, as an amateur free agent, and pitched from then through 2005 under the name of Jairo Garcia. During spring training in 2006 he revealed his real name to be Santiago Casilla, and that he had used false documents which listed him as three years younger than his actual age when he first signed in 2000.

Casilla pitched small parts of 2004, 2005, and 2006 for the Athletics as a reliever. In 2007, Casilla was recalled by the Athletics from Triple-A after going 2–1 with a 4.13 ERA with 29 strikeouts in 24 innings. He stepped into a bullpen that had Huston Street, Justin Duchscherer, and Kiko Calero on the disabled list. Casilla started off well, going 2–1 with 2 saves and a 0.45 ERA in his first 16 games. But he wound up finishing the season with a 3–1 record and a 4.44 ERA in 46 games. He pitched in the A’s bullpen in the 2008 and 2009 seasons with mixed success.

At the end of 2009, he was granted free agency by the A’s and signed a minor league contract with the San Francisco Giants for 2010. He made his Giants debut on May 21, 2010 against his former team in Oakland and remained with the team for the remainder of the season until they won the World Series. He returned to the Giants for 2011 as a member of the bullpen and took over as its closer after Brian Wilson went on the disabled list in August.

Casilla picked up his first career base hit, a bases loaded, two out RBI ground ball single between Diamondbacks first baseman Paul Goldschmidt and Aaron Hill on September 14, 2012. Casilla was the winning pitcher in Game 4 of the 2012 World Series as the Giants swept the Detroit Tigers.

On December 17, 2012, Casilla re-signed with the Giants for 3 years at $15 million. On May 28, 2013, Casilla underwent knee surgery to remove a cyst and was placed on the 15-day DL.

On May 17, 2015, in a 9–8 win over the Cincinnati Reds at Great American Ball Park, Casilla entered the bottom on the ninth inning and struck out three batters on nine pitches, thus recording an immaculate save. Casilla was the third San Francisco Giant in franchise history to pitch an immaculate inning, joining Trevor Wilson and Orel Hershiser.

On April 25, 2016, Casilla successfully logged his 100th save, closing 1 & 1/3 innings against the San Diego Padres. In 2016 he saved 31 games, but tied for the major league lead in blown saves, with nine.

On January 20, 2017, Casilla signed a two-year, $11 million contract with the Oakland Athletics. He served as the A’s closer to begin the season before being removed as closer. He was 16/23 in save opportunities. In 59 innings, he had an ERA of 4.27 with 57 strikeouts.

Casilla signed a minor league deal with Colorado on July 26 after being released by the Athletics on July 19. He elected free agency on November 3, 2018.