Mike Timlin was born in Midland, Texas to Jerome Francis Timlin Sr. and Nancy Sharon Beyer. Timlin graduated from Midland High School in Midland, Texas. He then attended and pitched at Southwestern University in Georgetown, Texas, where he was a member of Phi Delta Theta.

Timlin was known for his 93 mph fastball. His sliders and sinkers had a downward break, inducing a significant number of ground balls. Timlin assisted on the final out of 1992 World Series in Game 6 by fielding a bunt by Atlanta Braves outfielder Otis Nixon and throwing to Joe Carter at first base. Timlin also recorded a save for the game. Joe Carter gave Timlin the game ball shortly after the play was made.

In 2004, Timlin made 76 pitching appearances, the third-most in Red Sox history and tied for fourth in American League history. On September 3, he made his 800th major league appearance, becoming only the 29th pitcher in major league history to reach that mark. Timlin made his 1,000th career appearance on August 31, 2007, against the Baltimore Orioles.

Timlin's 2005 season exceeded expectations when he posted the best numbers of his career. In a career-high 81 appearances he recorded a career-low 2.24 ERA. Also worth noting, he allowed only two home runs in his 80 1⁄3 innings pitched.

After his first three seasons with the Red Sox, Timlin struggled with injuries to his right shoulder and left oblique in 2006 and the beginning of 2007, making multiple visits to the disabled list. In the period after his return from the disabled list through July 23, 2007, Timlin recorded over 14 consecutive scoreless innings and appeared to have overcome any shoulder issues.

Timlin appeared in the eighth inning of Game 1 of the 2007 World Series, pitching one inning of scoreless relief. The appearance came 15 years to the day that Timlin closed out the final game of the 1992 World SeriesAs of 2007, Timlin was the only active player to have played on four non-New York Yankees championship teams: (the 1992 and 1993 Toronto Blue Jays and the 2004 and 2007 Boston Red Sox).

On September 15, 2008, while playing the Tampa Bay Rays, Timlin set the record for most appearances by a right-handed relief pitcher with 1,050 games. He passed Kent Tekulve for first on the all-time list.

In 2008, his ERA rose to a career-high 5.66, giving up 1.6 home runs per 9 innings.  As of the end of the 2008 season, Timlin ranked seventh in appearances among all-time pitchers, with 1,058 appearances. He was second among active pitchers, behind only Mike Stanton (1,178).

On April 19, 2009, Timlin was honored by the Red Sox. He thew out the ceremonial first pitch on "Mike Timlin Day" at Fenway Park. On July 29, 2009, Timlin signed a minor league contract with the Colorado Rockies and made a brief appearance for the Casper Ghosts in Casper, Wyoming for the Rockies rookie league affiliate. On August 15, 2009 Timlin was released by the Colorado Rockies, following which he retired from baseball.