From 1995 to 2002 he worked at The Second City and IO Theater in Chicago, an experience that introduced him to 30 Rock creator Tina Fey and prepared him for his role on her show. McBrayer appeared in over 80 sketches on Late Night with Conan O’Brien (2002–04), often lampooning his own Southern upbringing by playing stereotypical hillbilly characters. On August 10, 2007, and September 20, 2008, he made cameo appearances on Late Night playing Kenneth Parcell from 30 Rock. During the first appearance, O’Brien remarked to him, “I thought you were above this now.” McBrayer later reprised his role as Kenneth twice (November 24, 2009, and January 13, 2010) after O’Brien’s switch to The Tonight Show. He appeared on O’Brien’s Legally Prohibited From Being Funny on Television Tour at stops in Eugene, Oregon, Universal City, California (on the Universal Studios lot where O’Brien taped Tonight), and the final tour stop in Atlanta; McBrayer is from Macon which is southeast of Atlanta and in Georgia.

In 2008, he was featured throughout the video for “Touch My Body” by Mariah Carey. McBrayer said he accidentally hit Carey in the face with a Frisbee during the shooting for the video. McBrayer also appears in the first episode in the fifth season of Tim and Eric Awesome Show, Great Job! as a spokesman in a spoof advertisement for the fictional “Diarrhea-phragm.” He worked as a voice actor in the American animated series Ugly Americans and in a recurring role on the second season of Phineas and Ferb.

In 2010, he played a role in Cats & Dogs: The Revenge of Kitty Galore. He regularly appears in the skit “Knock Knock Joke of the Day” on the hit children’s television show, Yo Gabba Gabba! since season 2 and stars in Craig McCracken’s new TV show Wander Over Yonder on the Disney Channel. In 2011, he also appeared in The Simpsons, in the season 22 episode, “The Great Simpsina” as Ewell Freestone, ‘Peach Guy’. In the fall of 2012, Jack lent his voice to the Disney animated film Wreck-It Ralph, playing the title character’s close friend and video game opponent Fix-It Felix, Jr. More recently, he has starred alongside Los Angeles Clippers player Blake Griffin in Kia commercials, and in 2013 appeared in a commercial for Barnes & Noble. McBrayer’s Adult Swim series The Jack and Triumph Show premiered in February 2015. In September 2016, on the season 10 premiere episode of The Big Bang Theory, McBrayer played Penny’s older brother Randall.

In 2018, McBrayer was one of the actors who voiced the audiobook A Day in the Life of Marlon Bundo.

In December 2018, it was announced McBrayer would make his West End stage debut in the transfer of the Broadway musical Waitress, playing the role of Ogie, until June 15 of 2019. The production opened at the Adelphi Theatre, London on February 8, 2019. In June 2019, he appeared on ITV’s The Sara Cox Show.

In 2020, McBrayer will recur on the comedy-mystery series Mapleworth Murders for Quibi.