Doolittle attended Shawnee High School in Medford, New Jersey, where he was a stand-out pitcher. Doolittle led Shawnee to a state championship. Doolittle played for the University of Virginia as both a starting pitcher and first baseman. He formerly held the record for wins in a career for a Virginia pitcher — 22 — which has since been passed by Danny Hultzen. In 2005, he played collegiate summer baseball with the Harwich Mariners of the Cape Cod Baseball League. In 2005 and 2006, Doolittle was named to the USA National (Collegiate) Baseball Team.

The Oakland Athletics selected Doolittle in the first round, with the 41st overall selection, in the 2007 Major League Baseball draft, as a first baseman and outfielder. He made his professional debut on June 18, 2007 and was expected to make his major league debut in 2009.

Despite being injured for most of the 2009 season, Doolittle was ranked tenth in Oakland’s farm system according to Baseball America. Doolittle missed the entire 2010 season while rehabbing from two knee surgeries. In the 2011 offseason, he was placed on Oakland’s 40-man roster to be protected from the Rule 5 draft. After missing more than two years, Doolittle converted back to pitching, making his professional pitching debut in the instructional league in Arizona in 2011.

After pitching just 26 professional innings, 25 of those at three minor league stops in 2012, Doolittle was called up to the majors on June 5, 2012, against the Texas Rangers pitching one and a third inning while striking out three with all fastballs and none going below 94 mph. He quickly became a key bullpen piece as the top lefty specialist earning his first career save on July 21 against the New York Yankees. He served as a set-up man for A’s closer Grant Balfour the rest of the way as Oakland went on to win the AL West on the final day of the season.

Doolittle signed a five-year, $10.5 million extension with the Athletics on April 18, 2014.

In November 2015, Doolittle and Dolan hosted a Thanksgiving dinner in Chicago for 17 Syrian refugee families. In October 2016, he was one of several professional athletes to denounce Republican presidential candidate Donald Trump’s comments about non-consensual groping of women as not being “locker room talk.” Doolittle identifies as independent politically. In 2019, the Washington Post reported that, because of several actions by the president, he was not going to attend a ceremony at the White House following his team winning the World Series.

Doolittle is active off the field with a number of charities and he was recognized for his work in 2016 by being nominated for the Roberto Clemente Award. Doolittle supports Operation Finally Home, a nonprofit dedicated to providing housing for U.S. military veterans and their families, and Swords to Ploughshares, a Bay Area organization devoted to helping veterans with housing and employment. In June 2015, when the Oakland Athletics Pride Night received backlash from some fans for the team’s support of LGBT rights, Doolittle and then-girlfriend Eireann Dolan bought hundreds of game tickets, which they donated to local LGBT groups, and raised an additional $40,000 in donations. Doolittle is an ally and LGBT rights activist.

While on rehab assignment with the Triple-A Nashville Sounds, Doolittle pitched the seventh inning of a combined no-hitter against the Omaha Storm Chasers on June 7, 2017. Starter Chris Smith pitched the first six innings and was then followed by Doolittle, Tucker Healy, and Simón Castro who each pitched one inning.

On July 16, 2017, Doolittle was traded to the Washington Nationals, along with Ryan Madson, for Blake Treinen, Sheldon Neuse, and Jesus Luzardo. On July 18, Doolittle recorded his first save for the Nationals in a 4-3 win over the Los Angeles Angels. In 30 games for the Nationals, he was 1-0 with a 2.40 ERA in 30.0 innings and was 21/22 in save opportunities.

Doolittle and Eireann Dolan married on October 2, 2017, eloping the day after the Washington Nationals’ last game of the regular season.

For the 2018 season, he was named closer to begin the season and up until July 11, he was 22/23 in save opportunities before falling to the disabled list with a left toe inflammation. He was activated off the disabled list on September 7.

In 2018, he was 3-3 with 25 saves (7th in the NL) and a 1.60 ERA, as in 43 relief appearances he pitched 45.0 innings and struck out 60 batters (12.0 per 9 innings). He threw a four-seam fastball 88.8% of the time, tops in MLB.

In 2019, he was 6-5 with 29 saves (6th in the NL) and a 4.05 ERA, as in 63 relief appearances he pitched 60.0 innings and struck out 66 batters. He led the NL in games finished (55), powering his Nationals to a World Series appearance and a save in Game 1. In 10 and a third innings during the postseason, he gave up only two runs and six hits while striking out eight.