As a young child, she appeared with her mother in Heartburn (credited under the name Natalie Stern to avoid press scrutiny). Twenty months old at the time of filming, she received a positive review in the New York Times. After graduating from college in 2005, she made her off-Broadway debut alongside Michael C. Hall in the premiere of Noah Haidle's Mr. Marmalade, for which she won a Theatre World Award. In 2007, she received a Lucille Lortel Award nomination for her performance in Theresa Rebeck's The Water's Edge.

Gummer made her motion-picture debut as an adult with a minor role in Lasse Hallström's The Hoax (2006), starring Richard Gere. In 2007, she starred with her mother in Michael Cunningham's film adaptation of Susan Minot's novel Evening, playing her mother as a young woman. The film, directed by Lajos Koltai, featured Vanessa Redgrave, Glenn Close, and Claire Danes. She portrayed Sally Adams in the 2008 HBO mini series John Adams, which details the life of the second President of the United States. She made her Broadway debut in the Tony Award–nominated revival of Les liaisons dangereuses in 2008, for which she received critical praise. She also guest-starred on the CBS legal drama The Good Wife in the season 1 episode "Bad" portraying Nancy Crozier. Her character returned again on season 2 episodes "Cleaning House" and "Getting Off".

After a year and a half of dating, Gummer became engaged to actor Benjamin Walker in October 2009. They married in July 2011 at her parents' home in Connecticut, and resided in Park Slope, Brooklyn, New York City. In March 2013, it was announced that Gummer and Walker had amicably separated, with Walker becoming engaged to his next wife in 2014.

In 2010, she starred in The Lightkeepers with Richard Dreyfuss, Tom Wisdom, Blythe Danner and Bruce Dern.

Starting in 2011, she starred in the ABC medical show Off the Map with Zach Gilford and Valerie Cruz. The show was created by Jenna Bans and Shonda Rhimes. In April 2011, she starred with Hamish Linklater and Alison Fraser, in the Off-Broadway production of The School For Lies written by David Ives, directed by Walter Bobbie, and produced by Classic Stage Company.

In 2012, she starred in the CW's Emily Owens, M.D. The series was canceled early during its first season, but the network continued to air the rest of its 13-episode order. Starting in 2015, she began a featured role in the WGN America series Manhattan.

From October to December 2015, she starred in the original production of the play Ugly Lies the Bone for the Roundabout Theater Company, and received a 2016 Drama Desk Award nomination for Outstanding Actress in a Play for her performance.

On February 1, 2016, it was announced that Gummer had joined the cast of the Amazon series The Collection as a series regular in the role of Helen Sabine.

Starting in 2018, she has been cast in the upcoming third season of HBO’s anthology drama True Detective. She’ll play Lucy Purcell, a mother of two children who finds herself involved in a crime.