Mulgrew's first television role was of older daughter Mary Ryan on the soap opera Ryan's Hope from 1975 to 1977. She became a fan favorite and is still associated with the show long after its cancellation.  Mulgrew remains friends with former co-star Ilene Kristen and presented a special Soap Opera Digest Award to Ryan's Hope creator Claire Labine in 1995.

Mulgrew left Ryan's Hope to take on the role of Kate Columbo in the short-lived 1970s series Mrs. Columbo (also known as Kate Loves a Mystery among other titles). Initially, Kate Columbo was the wife of Lt. Columbo, presumed to be the famed L.A.P.D. detective from Columbo, starring Peter Falk; however, revisions of the series during filming led to the storyline that there was another Lt. Columbo, and eventually Kate Columbo and he divorced, leading the character to assume the surname Callahan.

Among Mulgrew's television guest roles were Garnet McGee, a country singer, on Dallas; Hillary Wheaton, a Canadian anchorwoman wrestling with alcoholism, on Murphy Brown; and legal counselor Janet Eldridge, a romantic interest of Sam Malone, in the three-part fourth season finale of Cheers entitled "Strange Bedfellows."

In 1993, Mulgrew received an Honorary Doctorate from Seton Hall University for artistic contributions.

Mulgrew is perhaps best known for her role as Captain Kathryn Janeway on the science fiction television series Star Trek: Voyager which ran from 1995 to 2001. She was a last minute replacement for Canadian actress Geneviève Bujold who was cast as Captain Nicole Janeway but then dropped out after filming just a few scenes of the series' first episode, stating that a TV series work schedule was too demanding. The character's name was changed to Kathryn and history was made in the Star Trek franchise when she became the first female captain, as a series regular, in a leading role.

Something of a Katharine Hepburn look-alike, Mulgrew starred in a one-woman play called Tea at Five, a monologue reminiscence based on Hepburn's memoir Me: Stories of My Life. Tea at Five is also available as an audio recording.

In 2006 Mulgrew performed in The Exonerated at the Riverside Studios located in London, England. In the spring of 2007, she appeared in the NBC television series The Black Donnellys as Helen Donnelly. She also performed the lead role in an off Broadway production called Our Leading Lady written by Charles Busch. In the Fall of 2007, Mulgrew went on to grace the New York theatre stage, yet again, as Clytemnestra for Charles L. Mee's Iphigenia 2.0 (Signature Theatre Company).

Kate Mulgrew won the Saturn Award for "Best TV Actress" in 1998 and the Golden Satellite Award for "Best Actress in a TV series drama" in 1998. She was nominated for the Golden Globe Award for "Best Dramatic Actress" in 1979.

Mulgrew has also contributed her voice to various video games including reprising her role as Janeway in the video game Star Trek Legacy. Furthermore, she has voiced several audio books: Mosaics (Star Trek: Voyager) by Jeri Taylor, Miami, It's Murder by Edna Buchanan, and Everything to Gain by Barbara Taylor Bradford. Also, Mulgrew added her voice for projects with the Trappist monks of New Melleray Abbey as well as the MetroHealth System of Cleveland, Ohio.

Additionally, she contributed to a new book, Voices of Alzheimer's.