Born to well-to-do Jewish-American parents, Herman Radner and Henrietta Dworkin, in Detroit, Michigan, Radner graduated from the prestigious University-Liggett School in Grosse Pointe in 1964, and then studied drama at the University of Michigan, where she began her broadcasting career as the weather girl for college radio station WCBN. She moved to Toronto, Canada and had her first professional stage experience in a production of Godspell, after which she joined the Toronto Second City comedy troupe.

Radner was a featured player on the National Lampoon Radio Hour, a half-hour comedy program syndicated to some 600 U.S. radio stations from 1973 to 1975. Fellow cast members included John Belushi, Richard Belzer, Chevy Chase and Bill Murray.

She first rose to widespread fame as one of the original "Not Ready For Prime Time Players" on Saturday Night Live. (She was the first actor cast for the show.) Between 1975 and 1980 she created such characters as Roseanne Roseannadanna (a coarse woman with long black hair that always seemed to end up in places it didn't belong), Baba Wawa (a spoof of journalist Barbara Walters, exaggerating the latter's lisp), and Emily Litella, an elderly woman who gave angry and misinformed editorial replies (on topics such as "violins on television" and "protecting endangered feces") on the show's Weekend Update news segment. Once corrected on her misunderstanding, Litella would end her segment with a polite "Never mind" - or later on, she would answer Jane Curtin's frustration with her with a simple "Bitch!"

Radner projected an innocence into her lines that wouldn't have worked with other performers. "I guess in France, you don't order French fries," she said in one routine. "You just order fries. They'll know." Radner had a knack for combining extreme physical comedy with soft, caring characters that were easy to love. (There is a legend that Radner broke several ribs during one comedy sketch that required her to slam herself against a door repeatedly, but the next day she went on as scheduled.) Radner also battled bulimia during her time on the show (she once told a reporter that she had thrown up in every toilet in New York City), and had a relationship with co-star Bill Murray which ended badly. In 1979, incoming NBC President Fred Silverman offered Radner her own prime time variety show, which she ultimately turned down. That year, she was one of the hosts of the Music for UNICEF Concert at the United Nations General Assembly.

In her final season of Saturday Night Live, Radner appeared on Broadway in a successful one-woman show that featured racier material, such as the humorous song "Let's Talk Dirty to the Animals". This show was captured on film in 1981 as Gilda Live! and co-starred Paul Shaffer and Don Novello. The play was also released as an album recording -- the play was a qualified success, the film and album were failures.

She spent most of the next decade keeping a surprisingly low profile, aside from appearances in such films as Hanky Panky, The Lady in Red, and Haunted Honeymoon. In the late 1980s, she was diagnosed with ovarian cancer. Even with the support of her second husband, actor Gene Wilder, whom she had met while co-starring with him in Hanky Panky (she had previously been married to Saturday Night Live band leader G.E. Smith), she suffered extreme physical and emotional pain as a chemotherapy patient. Eventually, she was told she had gone into remission, and she wrote a memoir about her life and struggle with the illness, called It's Always Something. The book was written by Radner in tribute to cancer sufferers everywhere, and she used humor to overcome tragedy and pain. The book's title came from a common catch-phrase from her Saturday Night Live character Roseanne Roseannadanna, who would often quote an elderly relative by saying "It just goes to show ya...it's always something! If it's not one thing, it's another!"

In 1988 she guest-starred as herself on It's Garry Shandling's Show to great critical acclaim. (When Garry asked her why she had not been seen for awhile, Radner replied "Oh, I had cancer. What did you have?") She planned to host an episode of SNL that year but a writers' strike caused the cancellation of the rest of the season. She wanted to host the next year, but in 1989 doctors did a more detailed examination and discovered that Radner's cancerous cells had not all been removed and had spread to other areas of the body. She died in Cedars-Sinai Medical Center in Los Angeles, California, in 1989, where she had been admitted for a CAT scan. She was given a sedative and passed into a coma. After three days, she died without regaining consciousness, with Wilder at her side.

Wilder has since established the Gilda Radner Ovarian Detection Center at Cedars-Sinai to screen high-risk candidates (such as women of Ashkenazi Jewish descent) and run basic diagnostic tests. He testified before a Congressional committee that her condition was misdiagnosed and that if doctors had inquired more deeply into her family background they would have found numerous cases of ovarian cancer and might have attacked the disease earlier.

Wilder continued his involvement in both detection and treatment of ovarian cancer. In tribute to Radner, Gilda's Club was founded. It is a place where cancer patients and their families can go to be around other people in the same situation to share support, coping and wellness strategies. It grew to multiple locations across the country.

In 1992, Radner was inducted into the Michigan Women's Hall of Fame for her achievements in arts and entertainment. In 2002 the ABC television network aired a TV-movie about her life, starring fellow Midwestern Jewish American actress Jami Gertz.