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Poehler joined the cast of Saturday Night Live during the 2001–2002 season, her debut episode being the first one produced after the 9/11 attacks — with host Reese Witherspoon, musical guest Alicia Keys, and New York City Mayor Rudy Giuliani as a special guest. Poehler was promoted from featured player to full cast member in her first season on the show, making her the first woman and the third person to have earned this distinction (after Harry Shearer and Eddie Murphy).

Beginning with the 2004–2005 season, she co-anchored "Weekend Update" with Tina Fey, replacing Jimmy Fallon. In a TV Guide interview, Fey said that with Poehler co-anchoring, there is "double the sexual tension". When Fey left after the 2005–2006 season to devote time to the sitcom she created, 30 Rock, Seth Meyers joined Poehler at the anchor desk. In 2008, Poehler was nominated for the Primetime Emmy Award for Outstanding Supporting Actress in a Comedy Series, the first SNL cast member recognized in this category. She was heavily favored to win by many critics, but ultimately lost to Jean Smart of Samantha Who?. She was nominated once again in 2009, but lost to Kristin Chenoweth of Pushing Daisies. On September 13, 2008, the SNL season premiere opened with Fey and Poehler as Sarah Palin and Hillary Clinton, respectively, performing a joint political campaign spot.

It was announced on September 16, 2008, that Poehler would be leaving SNL in October to give birth to her first child. On the episode that aired on October 25, 2008, it was announced by "Weekend Update" co-anchor Seth Meyers, who anchored the segment alone, "Amy Poehler is not here because she is having a baby", to wild applause from the audience. At the end of "Weekend Update", special guest Maya Rudolph and cast member Kenan Thompson sang a custom rendition of "Can't Take My Eyes Off You" for Poehler, changing the words: "We love you Amy, and we just can't wait to meet your baby!" Meyers signed off: "For Weekend Update, I'm Seth Meyers — we love you Amy!"

Poehler returned to the show on November 3, 2008, during the "SNL Presidential Bash '08", "hosting" as Hillary Clinton. The Bash was pre-taped from scenes shot between September and October. Her return to SNL after her pregnancy was on December 6, 2008, where she stayed for two weeks. During "Weekend Update", on December 13, she thanked her family, friends, and fans for the continued support and announced that it would be her last show. On April 18, 2009, a Saturday Night Live special, "The Best of Amy Poehler", aired.[20] Poehler returned for "Weekend Update" (signing off with "...and he's Seth Meyers") and joined the "chorus" for Will Ferrell's "Goodnight Saigon" (along with the SNL cast and Tom Hanks, Maya Rudolph, Norm Macdonald, Paul Rudd, Artie Lange, Anne Hathaway, and Green Day) on the SNL season finale on May 16, 2009.