As a result of her marriage to Gene Roddenberry and her ongoing relationship with Star Trek – participating in some way in every series to date – she was sometimes referred to as "the First Lady of Star Trek". She and Gene Roddenberry were married in Japan on August 6, 1969, after the cancellation of the Star Trek: The Original Series. They had one son together, Eugene Rod Roddenberry Jr., born in 1974.

Hudec began taking acting classes as a child. She attended the University of Miami in Coral Gables, Florida, then had some stage roles and came to Hollywood. In the late 1950s and 1960s, she had bit parts in a few movies and small roles in TV series. She worked at the Desilu Studios on several TV shows, including Bonanza, The Untouchables, The Lucy Show, and The Lieutenant (produced by Gene Roddenberry). She received training in comedy from Lucille Ball. In 1960, she played Gwen Rutherford on Leave It to Beaver. She was also briefly seen in the film Will Success Spoil Rock Hunter? in an ad parody at the beginning of the film. Barrett is the mother of Eugene Wesley "Rod" Roddenberry, Jr.

In various roles, Barrett participated in every incarnation of the popular science fiction Star Trek franchise produced during her lifetime, including live-action and animated versions, television and cinema, and all of the time periods in which the various series have been set.

She first appeared in Star Trek's initial pilot, "The Cage" (1964), as the USS Enterprise's unnamed first officer, "Number One". Barrett was romantically involved with Roddenberry, whose marriage was on the verge of failing at the time, and the idea of having an otherwise unknown woman in a leading role because she was the producer's girlfriend is said to have infuriated NBC network executives who insisted that Roddenberry give the role to a man. William Shatner corroborated this in Star Trek Memories, and added that female viewers at test screenings hated the character as well. Shatner noted that women viewers felt she was "pushy" and "annoying" and also thought that "Number One shouldn't be trying so hard to fit in with the men." Barrett often joked that Roddenberry, given the choice between keeping Mr. Spock (whom the network also hated) or the woman character, "kept the Vulcan and married the woman, 'cause he didn't think Leonard [Nimoy] would have it the other way around."

Her role in subsequent episodes of Star Trek was altered to that of Nurse Christine Chapel, a frequently recurring character, known for her unrequited affection for the dispassionate Spock. In an early scene in Star Trek: The Motion Picture, viewers are informed that she has now become Doctor Chapel, a role which she reprised briefly in Star Trek IV: The Voyage Home. Barrett provided several voices for Star Trek: The Animated Series, including those of Nurse Chapel and a communications officer named M'Ress, an ailuroid officer who served alongside Uhura. She would return years later in Star Trek: The Next Generation, cast as the outrageously self-deterministic, iconoclastic Betazoid ambassador Lwaxana Troi, who appeared as a recurring character in the series. Her character often vexed the captain of the Enterprise, Jean Luc Picard, who spurned her amorous advances. Barrett later appeared as Ambassador Troi in several episodes of Star Trek: Deep Space Nine, where in stark contrast, she developed a strong relationship with Constable Odo.

She provided the regular voice of the onboard computers of Federation starships for Star Trek: The Original Series, Star Trek: The Next Generation, Star Trek: Deep Space Nine, Star Trek: Voyager, and most of the Star Trek movies. She reprised her role as a shipboard computer's voice in two episodes of the prequel series Star Trek: Enterprise. She also lent her voice to various computer games and software related to the franchise. Barrett had also made a point of attending a major Star Trek convention each year in an effort to inspire fans and keep the franchise alive.

Barrett is also one of six actors (the others being Jonathan Frakes, Kate Mulgrew, George Takei, Avery Brooks and Michael Dorn) to lend her voice to the CD-ROM Star Trek: Captain's Chair, reprising her role as the voice of the ships' computers.

On December 9, 2008, less than 10 days before her death, Roddenberry Productions announced that she would be providing the voice of the ship's computer once again, this time for the 2009 motion picture relaunch of Star Trek. Sean Rossall, a Roddenberry family spokesman, stated that she had already completed the voiceover work, approximately December 4, 2008. The film is dedicated to her as well as Gene.

Barrett and her husband, Gene Roddenberry, were honored in 2002 by the Space Foundation with the Douglas S. Morrow Public Outreach Award for their work creating awareness of and enthusiam for space.

She appeared as Primus Dominic in Roddenberry's 1973 post-apocalyptic TV drama pilot, Genesis II.