- She first appeared in Star Trek's initial pilot episode "The Cage" as the USS Enterprise's unnamed First Officer, "Number One". (According to the book Inside Star Trek: The Real Story by Herbert Solow and Bob Justman, Barrett was cast as Number One because she was romantically involved with Roddenberry at the time. Solow and Justman claim the network was furious that Roddenberry would attempt to put an unknown actress in such a prominent role simply because she was his girlfriend, and allege that Roddenberry later claimed that the network told him to remove her because they did not want a woman to be in such a high position of authority.)
- Her role was changed to that of Nurse Christine Chapel when Star Trek became a regular series; she continued this role in several of the subsequent Star Trek movies.
- She provided numerous voices for the animated Star Trek series.
- Years later she was cast as the outrageously determined and iconoclastic Betazoid Ambassador Lwaxana Troi, who was a recurring character in Star Trek: The Next Generation and who also appeared in some episodes of Star Trek: Deep Space Nine.
- She also provided the regular voice of starship onboard computers for the original Star Trek series, Star Trek: The Next Generation, Star Trek: Deep Space Nine, Star Trek: Voyager, and the Star Trek movies. She reprised her role as a shipboard computer voice in two episodes of the prequel series Star Trek: Enterprise that involved later time periods.
As a result of her providing the computer voice in the Enterprise episode "In a Mirror, Darkly", Barrett became the first and only actor to participate in all six Star Trek series as well as the both the TOS and TNG-era film series.
Barrett also played the character Dr. Julianne Belman in Earth: Final Conflict, which was also created by Gene Roddenberry. She made a guest appearance in a Babylon 5 episode "Point of No Return", as Lady Morella, the psychic widow of the Centauri emperor; her role foreshadowed major plot elements in the series. Parodying her voice work as the computer for the Star Trek series, Majel performed as a guest voice on Family Guy as the voice of Stewie's ship.