Gasteyer developed comedy experience with the famed Los Angeles improv – sketch comedy group The Groundlings. Prior to joining SNL, she had small roles on Seinfeld (where she was a doomed customer of The Soup Nazi), as well as on the shows Party of Five, Hope & Gloria and NYPD Blue.

Gasteyer joined the cast of Saturday Night Live beginning in 1996 for 6 seasons.

Gasteyer left SNL in 2002. Since then, she has appeared in various television programs and films as well as in stage productions. In 2004, Gasteyer played the mother of the main character Cady Heron (Lindsay Lohan) in the feature film Mean Girls, written by SNL castmate Tina Fey. The film also featured other former SNL castmates, including Fey, Tim Meadows, and Amy Poehler. She also appeared in the 2005 Showtime movie musical Reefer Madness as Mae, the doomed girlfriend of Jack and owner of the Reefer Den.

Gasteyer starred in a revival of The Threepenny Opera on Broadway as Mrs. Peachum, along with Jim Dale, Alan Cumming, and Cyndi Lauper. The production ran from March 24 through June 25, 2006.

On June 24, 2005, Ana began originating the role of Elphaba in the Chicago sit-down production of the musical Wicked. The production opened July 13, 2005, and Gasteyer was later nominated for a Jefferson Award for her portrayal. She played her final performance on January 22, 2006, and was replaced by her standby Kristy Cates. She later reprised the role in the Broadway production from October 10, 2006, through January 7, 2007.

On April 12, 2007, it was announced that Gasteyer has joined the cast of the new musical, Writing Arthur, for the 2007 New Works Festival for TheatreWorks in California which ran from April 14–22.

Gasteyer performed in Chicago once again in the Stephen Sondheim musical Passion at Chicago Shakespeare Theater from October 2, 2007 to November 11, 2007.

She was originally cast as Gloria in the Encores! Summer Series production of Damn Yankees, but was replaced by Megan Lawrence during rehearsals due to injury.

Gasteyer took part in the reading of the Broadway musical version of “The First Wives Club.”

She most recently starred as Kitty Dean in the Broadway play, The Royal Family which began performances at the Samuel J. Friedman Theatre on September 15, 2009 and officially opened on October 8, 2009. The show concluded its run on December 13, 2009.

Gasteyer also starred as Frisco Kate Fothergill in the City Centers Encores! production of Girl Crazy, running from November 19–22, 2009.

She returned to Saturday Night Live for a special Mother's Day episode on May 8, 2010.