Johnston was born and raised in Chicago. He attended Columbia College Chicago, where he studied acting and comedy alongside other future Mr. Show alumni such as Dino Stamatopoulos, Scott Adsit and Eric Hoffman. After graduating college, Johnston later joined the Chicago Second City's touring comedy for a number of years and also performed in stage shows at the Annoyance Theater in Chicago. Jay Johnston's contribution to Mr. Show were generally of the "evergreen" variety, dealing with more or less universal themes as opposed to the more political and socially satirical material that his fellow writers and performers contributed to the series. As such, Jay was responsible for many of the show's memorable sketches and characters. Johnston often incorporated physical comedy into the show, most notably in "The Story of Everest" sketch, which features Johnston performing six identical backwards pratfalls into furniture. Since the conclusion of Mr. Show, Johnston has made guest appearances in a number of television programs such as Arrested Development, Community, Curb Your Enthusiasm, and Parks and Recreation, as well as appearing in films such as and Tenacious D in The Pick of Destiny. In 2006, Johnston regularly appeared as "The Boss" on FUEL TV's American Misfits, in the show's intro and outro sketch segments. The show is a half-hour mix of skateboarding and sketches, very much in the mold of the early 1990s skateboarding videos. Johnston starred on all three seasons of Comedy Central's The Sarah Silverman Program as "Officer Jay McPherson", the love interest of Sarah's sister Laura in the series. The series reunited Johnston with Mr. Show cast members Brian Posehn, Scott Aukerman, B. J. Porter, and Paul F. Tompkins. In 2011, Johnston appeared as a sketch regular in season two of the Comedy Central sketch comedy series Nick Swardson's Pretend Time. Johnston and fellow Mr. Show regulars Dino Stamatopoulos and Scott Adsit, were part of the creative team behind Adult Swim'sstop-motion animation series Moral Orel, which ran from 2005 to 2008. In 2010, Johnston provided voices for Stamatopoulos' Adult Swim series, Mary Shelley's Frankenhole. Johnston currently has a recurring voice role as Jimmy Pesto, Sr. in the FOXanimated seriesBob's Burgers.
