Radner graduated from Liggett and enrolled at the University of Michigan at Ann Arbor in 1964. While at the university, she made a lifelong platonic friend of fellow student David Saltman, who wrote a biography of her after her death. Saltman and his girlfriend took Radner along on a trip to Paris in the summer of 1966. According to Saltman, he was so affectionate with his girlfriend that they left Radner to fend for herself during much of their sightseeing. Radner was nervous and upset about gaining weight from the French cuisine, but Saltman paid little attention at the time.

Twenty years later, when details of Radner's eating disorder were reported in a book about Saturday Night Live by Doug Hill and Jeff Weingrad, Saltman had a strong emotional reaction. He realized that in 1966 she had been unable to discuss with anyone her eating disorder and its impact on their trip to France. (The book by Hill and Weingrad was published and received much media coverage during a period when Radner was consulting various doctors in Los Angeles about her symptoms of illness that turned out to be cancer.)

In Ann Arbor, Radner dropped out in her senior year to follow her boyfriend, Canadian Sculptor Jeffrey Rubinoff, to Toronto, where she made her professional acting debut in the 1972 production of Godspell with Future stars Eugene Levy, Andrea Martin, Victor Garber, Martin Short, and Paul Shaffer. Afterward, Radner joined The Second City comedy troupe in Toronto.

Radner was a featured player on the National Lampoon Radio Hour, a comedy program syndicated to some 600 U.S. radio stations from 1974 to 1975. Fellow cast members included John Belushi, Chevy Chase, Richard Belzer, Bill Murray, Brian Doyle-Murray, and Rhonda Coullet.

Radner gained name recognition as one of the original "Not Ready for Prime Time Players", the freshman group on the first (1975) season of Saturday Night Live. She was the first performer cast for the show, co-wrote much of the material that she performed, and collaborated with Alan Zweibel (of the show's writing staff) on sketches that highlighted her recurring characters. Between 1975 and 1980, she created characters such as obnoxious personal advice expert Roseanne Roseannadanna and "Baba Wawa", a parody of Barbara Walters. After Radner's death, Walters stated in an interview that Radner was the "first person to make fun of news anchors, now it's done all the time." Radner's lampooning of news anchors did owe something to Monty Python, however, which started doing that several years before Saturday Night Live.

Radner won an Emmy Award for "Outstanding Continuing or Single Performance by a Supporting Actress in Variety or Music" for her performance on Saturday Night Live in 1977. She posthumously won a Grammy Award for "Best Spoken Word Or Non-Musical Recording" in 1990.

News of her death broke as Steve Martin was rehearsing to act as the guest host for that night's season finale of Saturday Night Live. The show's performers and crew, including Lorne Michaels, Phil Hartman, and Mike Myers (who had, in his own words, "fallen in love" with Radner after playing her son in a BC Hydro commercial on Canadian television and considered her the reason he wanted to be on SNL), had not known how grave her situation was. Martin's planned opening monologue was scrapped; in its place Martin, in tears, introduced a video clip of a 1978 Sketch in which he and Radner had parodied Fred Astaire and Cyd Charisse in a well-known dance routine from The Band Wagon (1953). After the clip, Martin said it reminded him of "how great she was and of how young I looked. Gilda, we miss you."

In 1979, Radner appeared on Broadway in the successful one-woman show, Gilda Radner - Live From New York. The show featured material that was racier than NBC censors allowed on Saturday Night Live, such as the song "Let's Talk Dirty to the Animals". In 1979, shortly before Radner's final season on Saturday Night Live, her Broadway show was filmed by Mike Nichols under the title Gilda Live, co-starring Paul Shaffer and Don Novello, and the movie was released in theaters nationwide in 1980, with poor results. A Soundtrack album was also unsuccessful. During the Broadway production, Radner met her first husband, G. E. Smith, a musician who worked on the show. They were married in a civil ceremony in 1980.

In the fall of 1980, after all original SNL cast members departed from the show, Radner began working with actor Sam Waterston in the Jean Kerr play, Lunch Hour. They played two people whose spouses are having an affair, and as a reaction they start their own relationship consisting of trysts on their lunch hour. The show ran for more than seven months in various theaters in the United States, including the John F. Kennedy Center for the Performing Arts in Washington, DC. Newspaper critics, including Tom Shales, praised the play and Radner's performance in it.

Radner met actor Gene Wilder on the set of the Sidney Poitier film Hanky Panky (released in 1982), when the two worked together making the film. She described their first meeting as "love at first sight". She was unable to resist her attraction to Wilder as her marriage to Guitarist G. E. Smith deteriorated. Radner went on to make a second film with Wilder, The Woman in Red (released in 1984), and their relationship grew. The two were married on September 18, 1984, in Saint-Tropez. The pair made a third film together, Haunted Honeymoon (released in 1986) and remained married until her death in 1989.

In 1985, after experiencing severe fatigue and suffering from pain in her upper legs on the set of Haunted Honeymoon in the United Kingdom, Radner sought medical treatment. For a period of 10 months, various doctors, most of them in Los Angeles, gave her several diagnoses that all turned out to be wrong as she continued to experience pain.

Finally, on October 21, 1986, Radner was diagnosed with stage IV ovarian cancer. "She immediately underwent surgery and had a hysterectomy," wrote Jenny Song in a 2009 magazine article published by the American Association for Cancer Research. On October 26, "surgeons removed a grapefruit-size tumor from her abdomen," the article continued. Radner then began chemotherapy and radiation therapy treatment, as she wrote in It's Always Something, and the treatment caused extreme physical and emotional pain.

Four months after her ordeal with the National Enquirer, Radner saw her Saturday Night Live castmates one last time at Laraine Newman's 35th birthday party (in March 1987). According to Bill Murray when he heard she was leaving the party, he and Dan Aykroyd carried her around the Los Angeles house where the party was held, repeatedly saying goodbye to everyone. Since all the guests were comedians, they all did comedy bits with her repeatedly.

After Radner was told that she had gone into remission, she wrote It's Always Something (a catchphrase of her character Roseanne Roseannadanna's), which included details of her struggle with the illness. Life did a March 1988 cover story on her illness, titled "Gilda Radner's Answer to Cancer: Healing the Body with Mind and Heart." In 1988, Radner guest-starred on It's Garry Shandling's Show on Fox TV, to critical acclaim. When Shandling asked her why she had not been seen in public for a while, she replied, "Oh, I had cancer. What did you have?" Shandling's reply: "A very bad series of career moves ... which, by the way, there's no treatment for whatsoever." She repeated on-camera Mark Twain's apocryphal saying, "Reports of my death are greatly exaggerated." Radner planned to host an episode of Saturday Night Live that year, but a writers' strike caused the delay of the network television season.

In September 1988, after tests showed no signs of cancer, Radner went on a maintenance chemotherapy treatment to prolong her remission, but three months later, in December, she learned the cancer had returned. She was admitted to Cedars-Sinai Medical Center in Los Angeles on May 17, 1989, to undergo a CT scan. She was given a sedative and went into a coma during the scan. She did not regain consciousness and died three days later, from ovarian cancer and complications due to intestinal perforation on May 20, 1989; Wilder was at her side.

In 1991, Gilda's Club, a network of affiliate clubhouses where people living with cancer, their friends, and families, can meet to learn how to live with cancer, was founded by Joanna Bull, Radner's cancer psychotherapist, along with Radner's widower, Gene Wilder (also a cancer survivor) and broadcaster Joel Siegel (who later died after a long battle with cancer). The first club opened in New York City in 1995. The organization took its name from Radner's comment that cancer gave her "membership to an elite club I'd rather not belong to". Radner's story can be read in her book, It's Always Something.

Radner's death helped raise awareness of early detection of ovarian cancer and the connection to familial epidemiology. The media attention in the two years after Radner's death led to registry of 450 families with familial ovarian cancer at the Familial Ovarian Cancer Registry, a research database registry at the Roswell Park Cancer Institute in Buffalo, New York. The registry was later renamed the Gilda Radner Familial Ovarian Cancer Registry (GRFOCR). In 1996, Gene Wilder and Registry founder Steven Piver, one of Radner's medical consultants, published Gilda's Disease: Sharing Personal Experiences and a Medical Perspective on Ovarian Cancer.

In 2002, the ABC television network aired a television movie about her life: Gilda Radner: It's Always Something, starring Jami Gertz as Radner.

In 1992, Radner was inducted into the Michigan Women's Hall of Fame for her achievements in arts and entertainment. Through the generosity of many who participated in the 2002 ABC special, "Gilda Radner's Greatest Moments," (including Lynda Carter, Victor Garber, Eric Idle, David Letterman, Eugene Levy, Peter Mann, Steve Martin, Mike Myers, Paul Shaffer, Lily Tomlin and The Jim Henson Company), funds were raised to get Gilda a posthumous star on the Hollywood Walk of Fame. On June 27, 2003, she received a star on the Hollywood Walk of Fame at 6801 Hollywood Blvd. "Saturday Night Live" alumna Molly Shannon (and the host of the ABC special) served as Master of Ceremonies at the induction at which Laraine Newman, Gilda's Club founder Joanna Bull and Gilda's brother Michael F. Radner also spoke to dedicate the honor.

In 2007, Radner was featured in Making Trouble, a film tribute to female Jewish comedians, produced by the Jewish Women's Archive. Radner makes two comic book appearances. DC Comics Young Love #122 in 1976 and Marvel Team-Up #74 from 1978.

Many Gilda's Clubs have opened across the United States and in Canada. In July 2009, Gilda's Club Worldwide merged with The Wellness Community, another established cancer support organization to create the Cancer Support Community (CSC), which was legally adopted in 2011. As of 2012, more than 20 local affiliates of Gilda's Club were active. Although some local affiliates of Gilda's Club and The Wellness Community have retained their names, many affiliates have adopted the name Cancer Support Community following the merger.