Birbiglia was born in Shrewsbury, Massachusetts, the son of Mary Jean (née McKenzie) and Vincent Paul Birbiglia. He is the youngest of four children. He is of part Italian descent. Birbiglia attended St. John's High School, Shrewsbury, Massachusetts for one year, and graduated from St. Mark's School, Southborough, Massachusetts in 1996. He says seeing Comedian Steven Wright perform inspired him to begin writing jokes at age 16. He subsequently attended Georgetown University in Washington, D.C., graduating in 2000. During college he performed as a member of the Georgetown Players Improv Troupe, worked as a server in a comedy club, and began performing at The Improv in Washington, D.C.

Birbiglia moved to New York in 2000, and appeared on the Late Show with David Letterman in 2002. He told his first story on stage at the U.S. Comedy Arts Festival in Aspen, in 2003, and eventually became a semi-regular contributor to The Moth storytelling series. He released his first album, Dog Years, in 2004, followed by My Secret Public Album, Volume 1a (2005) a compilation of his appearances on The Bob and Tom Show. His next two albums, Two Drink Mike (2006), and My Secret Public Journal Live (2007) were released on Comedy Central Records. My Secret Public Journal Live was named one of the best comedy albums of the decade by The A.V. Club

In 2007 Birbiglia starred in the independent feature film Stanley Cuba, which debuted at the Santa Barbara International Film Festival and played in the Brooklyn and Milan film festivals. In 2008 CBS picked up a sitcom pilot loosely based on Birbiglia's life and featuring himself, Bob Odenkirk and Frances Conroy that ultimately never aired. He later called the failure of his pilot "the luckiest thing that ever happened to me."

In 2008, Birbiglia opened Sleepwalk with Me, a one-man show that straddled standup comedy and theater, off-Broadway at the Bleecker Street Theater. The show was presented by Nathan Lane, and the New York Times called it "simply perfect". Time Out New York named it their "Show of the Year" in 2009.

Birbiglia adapted his material into the 2010 book Sleepwalk with Me, and Other Painfully True Stories and the 2011 album Sleepwalk With Me Live. The book debuted at #29 on the hardcover nonfiction New York Times Bestseller List and number four on the hardcover nonfiction section of the Washington Post Political Bookworm Best Sellers. The book was also a finalist for the 2011 Thurber Prize for American Humor. Sleepwalk With Me Live debuted at #1 on the Billboard Comedy Charts.

Birbiglia made his directorial debut with Sleepwalk with Me, based on his one-man show and which he wrote, directed, and starred in. The film premiered at the 2012 Sundance Film Festival and won the NEXT Audience Award. It was selected in the "Festival Favorites" category at the South by Southwest film festival in Austin, Texas and also appeared in the Nantucket Film Festival where it won the award for best writer/director. The film was produced by Jacob Jaffke and This American Life's Ira Glass and stars Birbiglia, Lauren Ambrose, Carol Kane, James Rebhorn, and Cristin Milioti and has cameos by Glass, Kristen Schaal, Wyatt Cenac, David Wain, Jessi Klein, John Lutz and Marc Maron.

In 2011, Birbiglia mounted his second one-man show, My Girlfriend's Boyfriend, which ran for four months off-Broadway and won the Lucille Lortel Award for Outstanding Solo Show. He toured My Girlfriend's Boyfriend around the world with tour dates in the United States, Australia, Canada and the United Kingdom. On June 2, 2013, Birbiglia performed it for the last time at Carnegie Hall. My Girlfriend's Boyfriend was featured as the number one stand-up special of 2013 by Vulture, Paste, The Laugh Button and Laughspin. It was also named one of Time Out New York's Best Comedy Specials of 2013. Flavorwire listed the special as one of the top 20 funniest stand-up specials of all time.

For his second film, Birbiglia's drew upon his early experience with improv in Don't Think Twice (2016), which centers on a fictional Manhattan improv troupe. Birbligia also stars among an ensemble cast including Keegan-Michael Key, Gillian Jacobs, Kate Micucci, Tami Sagher, and Chris Gethard. The film was nominated for several awards, including the Critics' Choice Award for Best Comedy and St. Louis Film Critics Association award for Best Comedy.

Birbiglia's Thank God for Jokes comedy special was released on Netflix on February 28, 2017.