Margulies made her feature film debut in Steven Seagal's film Out for Justice , playing a prostitute looking to go straight. In 1994, Margulies was cast in the pilot episode of the NBC medical drama ER as Carol Hathaway, an emergency care nurse who, despondent over her relationship with pediatrician Doug Ross (George Clooney), attempted suicide. Her character was originally intended to die; however, the producers changed the plot and she remained on the show for six seasons until 2000. She won an Emmy Award for Best Supporting Actress in a Television Drama in 1994, and continued to be nominated for this award every year during her tenure on ER. She was the only series regular cast member to win an Emmy Award.

In March 2012, Margulies broke her silence on her decision to leave ER at the height of her career and a $27 million paycheck. She revealed that although everyone said it would be "career suicide" not to return to the medical drama she had starred in for six years, she was never concerned about the consequences.

"I'm a smart girl," said Margulies. "I had a year's worth of work waiting and a mortgage completely paid at age 32. I was under no illusion that I was going to be some big movie star. My dad said, 'If you got hit by a bus tomorrow, were you living your life truthfully, or were you waiting to get rich?' If I died and my soul started leaving my body, would I be looking down going, 'You idiot. You could have gone to Prague, you could have been on Broadway'? Those are the things I wanted to do."

After leaving the series, Margulies worked on stage and screen. On stage, she appeared in a MCC Theater production of Kate Robin's Intrigue With Faye, a Lincoln Center production of Jon Robin Baitz's Ten Unknowns, and The Vagina Monologues. Her film work after ER included Evelyn with Pierce Brosnan and Ghost Ship with Gabriel Byrne and Ron Eldard. She starred as the protagonist and narrator (Morgaine) in the TNT miniseries The Mists of Avalon and participated in the documentary film Searching for Debra Winger.

In 2004, she guest-starred in a two-episode arc in season 4 of the hit TV show Scrubs as Neena Broderick, an unscrupulous lawyer who sues Turk and has a brief sexual relationship with J.D. She starred in another miniseries on TNT, The Grid. In April 2006, she appeared in four episodes of the sixth season of The Sopranos, portraying realtor Julianna Skiff. In August 2006, she appeared in Snakes on a Plane as flight attendant Claire Miller. In December 2006, she played Jennifer Bloom in the Syfy Channel miniseries The Lost Room.

In an August 2006 interview with tvguide.com, Margulies said she was close to accepting an offer to return to ER for a four-episode arc, with Noah Wyle, that filmed in Hawaii during the 2005/06 season. However, she decided against it at the last minute. Margulies was invited to return during ER's final season, but the actress initially turned down the offer, saying she felt like she left Carol Hathaway in the perfect place and could not imagine bettering her departure episode. However, Margulies did return to ER for one episode during its 15th and final season.

She had a minor role in The Darwin Awards. In 2008, Margulies starred in the legal series Canterbury's Law, a Fox mid-season replacement show. She played the title character, Elizabeth Canterbury, a lawyer described as a "tough-minded defense attorney who isn't afraid to push boundaries in order to protect innocent clients." She was also credited as a producer of the show. The series was severely affected by the 2007–2008 Writers Guild of America strike; it was cancelled by the network after only six episodes.

In 2009, Margulies began starring in the CBS legal drama The Good Wife. She played Alicia Florrick, an attorney returning to legal practice after her husband Peter Florrick (played by Chris Noth) resigned as Illinois State's Attorney amid a sex and corruption scandal.

Since 2011, starting with the third season, Margulies was credited as a producer of The Good Wife. In 2013, the actress did the voice-over for a Chase credit card commercial. In 2014, Margulies narrated "Women in Business", an episode of season 2 of Makers: Women Who Make America.

In 2015, TIME featured Margulies as one of the "100 Most Influential People in the World." It noted her work to get Erin's Law, concerned with child sexual abuse, passed in every US state.

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