
Lane's maternal grandmother, Eleanor Scott, was a three-times married Pentecostal preacher of the Apostolic denomination, and Lane was influenced by the theatricality of her grandmother's sermons. Lane began acting professionally at the age of six at the La MaMa Experimental Theater Club in New York, where she appeared in an production of Medea. At 12 she had a role in Joseph Papp's production of The Cherry Orchard with Meryl Streep. Also at this time, Lane was enrolled in an accelerated program at Hunter College High School and was put on notice when her grades suffered from her busy schedule. At 13 years old, she turned down a role in Runaways on Broadway to make her feature film debut opposite Laurence Olivier in A Little Romance. At 14 years old, Lane was featured on the cover of Time, which declared her one of Hollywood's "Whiz Kids."

However, the two films that could have catapulted her to star status, Streets of Fire (she turned down Splash and Risky Business for this film) and The Cotton Club, were both commercial and critical failures, and her career languished as a result. After The Cotton Club, Lane dropped out of the movie business and lived with her mother in Georgia. According to the actress, "I hadn't been close to my mom for a long time, so we had a lot of homework to do. We had to repair our relationship because I wanted my mother back".
Lane returned to the business to make The Big Town and Lady Beware, but it was not until 1989's popular and critically acclaimed TV mini-series Lonesome Dove that Lane made another big impression on a sizable audience. She was nominated for an Emmy Award for the role. She also enjoyed positive reviews for her performance in the independent film My New Gun, which was well received at the Cannes Film Festival. She went on to appear as actress Paulette Goddard in Sir Richard Attenborough's big-budget biopic of Charles Chaplin, Chaplin.

In 2002, Lane starred in Unfaithful, a drama film directed by Adrian Lyne adapted from the French film The Unfaithful Wife. Lane played a housewife who indulges in an adulterous fling with a mysterious book dealer. The film featured several sex scenes. Lyne's repeated takes for these scenes were very demanding for the actors involved, especially for Lane, who had to be emotionally and physically fit for the duration. Unfaithful received mostly mixed to negative reviews, though Lane earned widespread praise for her performance. Entertainment Weekly critic Owen Gleiberman said, "Lane, in the most urgent performance of her career, is a revelation. The play of lust, romance, degradation, and guilt on her face is the movie's real story". She followed that film up with Under the Tuscan Sun, based on the best-selling book by Frances Mayes.

In 2008, Lane expressed frustration with being typecast and is "gunning for something that's not so sympathetic. I need to be a bitch, and I need to be in a comedy. I've decided. No more Miss Nice Guy". The actress has even contemplated quitting acting and spending more time with her family if she is unable to get these kinds of roles. She said in an interview, "I can't do anything official. My agents won't let me. Between you and me, I don't have anything else coming out".
In 2009, it was announced that Lane will star in Secretariat, a Disney film about the relationship between the 1973 Triple Crown-winning racehorse and his owner, Penny Chenery, whom the actress will be portraying.