She made her directorial film debut with Pitch Perfect 2 (2015), whose $69 million opening-weekend gross set a record for a first-time director.

She has appeared in films such as Wet Hot American Summer (2001), Sam Raimi's Spider-Man trilogy (2002–2007), Seabiscuit (2003), The 40-Year-Old Virgin (2005), Slither (2006), Invincible (2006), Zack and Miri Make a Porno (2008), Role Models (2008), The Next Three Days (2010), Man on a Ledge (2012), What to Expect When You're Expecting (2012), Movie 43 (2013), The Lego Movie film series (2014–2019), Love & Mercy (2014), Walk of Shame (2014), Magic Mike XXL (2015), Power Rangers (2017), Brightburn (2019), Call Jane (2022), Migration (2023), and Skincare (2024).

On television, Banks had a recurring role as Avery Jessup on the NBC sitcom 30 Rock (2010–2012), which earned her two Primetime Emmy Award nominations.

Taking the role would have required her to quit her education at the American Conservatory Theater, and as she had taken out student loans to complete her degree, she declined the offer.

[18] In August 2005, at the Williamstown Theatre Festival, she starred in William Inge's Bus Stop as Cherie, the aspiring nightclub singer.

"[20] In 2005, she appeared on the series Stella, and in May 2006, she had a role in the season five finale of the NBC sitcom Scrubs as Dr. Kim Briggs, the love interest of J.D.

[23] In 2007, Banks played the female lead in the comedy film Meet Bill, alongside Aaron Eckhart and Jessica Alba.

[21] That same year, she had a small role as Santa's little helper, Charlyne, in the Christmas comedy film Fred Claus, co-starring Vince Vaughn and Paul Giamatti.

In 2008, she played the ex-wife of Ryan Reynolds’ character and mother to his daughter in the comedy film Definitely, Maybe alongside Isla Fisher and Rachel Weisz,[24] starred with Seth Rogen as the eponymous female lead in the Kevin Smith comedy Zack and Miri Make a Porno,[21] and played United States First Lady Laura Bush in W., Oliver Stone's biopic of George W.

[27] Banks is a frequent co-star of actor Paul Rudd, the two having appeared in five films together to date (Wet Hot American Summer, The Baxter, The 40-Year-Old Virgin, Role Models, and Our Idiot Brother).

[29] Banks was cast as a love interest for Jack Donaghy (Alec Baldwin) in the fourth season of the Emmy Award-winning sitcom 30 Rock.

[21] Banks co-starred in the 2014 movie Every Secret Thing, playing Detective Nancy Porter investigating the disappearance of a young child with similarities to a case she had previously been involved with.

[48] Banks directed, produced, wrote, and starred as Bosley in the action comedy film Charlie's Angels, which was released in November 2019.

[50] In November 2019, it was announced that Banks was set to star in, direct, and produce a new adaptation of The Invisible Woman (1940), based on her own original story pitch.

[52] In June 2020, it was announced Banks would star as Ms. Frizzle in a live-action/animated hybrid of The Magic School Bus, which she will also produce through her company Brownstone Productions.

[53][54] Banks directed the 2023 comedy horror film Cocaine Bear, which she produced alongside Max Handelman for Brownstone Productions, and Phil Lord and Christopher Miller.

[55] In May 2021, Peacock announced that Banks would direct and star in the upcoming television series Red Queen, based on the dystopian novel of the same name.

[66] Banks was a vocal supporter of Hillary Clinton's 2016 presidential campaign and was involved in a rendition of Rachel Platten's single "Fight Song" with other celebrities at the 2016 Democratic National Convention.

[69] In September 2020, Banks urged her Instagram followers to contact VoteRiders to get information and assistance with voter ID requirements for the presidential election.