[6] Helgenberger began as a nightly weathergirl at KHGI-TV, the ABC affiliate in Kearney, while attending college (her name was changed by the producer to Margi McCarty.

After portraying the role of Blanche Dubois in a university production of A Streetcar Named Desire, she developed an interest in acting.

[7] While performing in a summer 1981 NU campus production of Shakespeare's Taming of the Shrew, where she played Kate, Helgenberger was spotted by a scout for the TV soap opera Ryan's Hope.

She also played a regular role as Natalie Thayer, opposite Margot Kidder and James Read, on the six-episode drama comedy series Shell Game (1987).

In 1989, Helgenberger made her feature-film debut in a leading role as an all-night answering-service operator in one segment of the Wheat brothers' horror anthology After Midnight.

She followed it up with a role in Steven Spielberg's romantic comedy-drama Always (starring Richard Dreyfuss, Holly Hunter, and John Goodman), a modern version of the original 1943 Victor Fleming film A Guy Named Joe.

During the early to mid-1990s, Helgenberger played the love interest to Woody Harrelson's character in The Cowboy Way (1994), and had a small role as Capt.

After playing a recurring role as George Clooney's love interest on NBC's medical drama ER, Helgenberger appeared as David Caruso's sex-starved widow on Showtime's Elmore Leonard's Gold Coast.

Starting in October 2000, Helgenberger starred in the role of Catherine Willows, a former show girl employed as a blood-spatter analyst, on the hit CBS drama CSI: Crime Scene Investigation.

In 2005, she and her fellow cast members won the Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Drama Series.

When CSI first started filming, Helgenberger visited the Clark County Coroner's Office to learn about her role, even viewing autopsies in progress.

She also starred as Dennis Quaid's wife and Scarlett Johansson's mother in writer-director Paul Weitz's romantic drama comedy In Good Company (2004).

[11] In December 2009, Forbes magazine placed Helgenberger third among primetime's top-earning women, with estimated earnings of $9.5 million, after Tyra Banks and Katherine Heigl.

[20] In May–June 2014, Helgenberger acted onstage in a production of the 2011 play The Other Place by Sharr White, put on by the Barrington Stage Company in Pittsfield, Massachusetts.

[22] In 2022, Helgenberger became a series regular on CSI: Vegas, joining the show in its second season and reprising her long-running role of Catherine Willows.