While working toward a career in roofing, Mewes made his film debut in Smith's 1994 film Clerks as Jay. The film was a huge success and ever since, Mewes has been closely identified with the role, which he also played in Mallrats (1995), Chasing Amy (1997), Dogma (1999), Jay and Silent Bob Strike Back (2001), Clerks II (2006), and the upcoming sequels Mallbrats (2016) and Clerks III (TBA). He also supplied Jay's voice in the short-lived Clerks: The Animated Series.

He, along with Kevin Smith, performed in the films Scream 3 and Bottoms Up and on Canadian television series Degrassi: The Next Generation. He appeared in two episodes of a three-part story arc in which he and Smith film a new addition to Smith's View Askew films titled Jay and Silent Bob Go Canadian, Eh? at Degrassi Community School. They appear again on Degrassi (in a two-part episode) at the premiere of Jay and Silent Bob go Canadian, Eh? in Canada. Mewes, Smith, and Degrassi star Stacie Mistysyn made the cover of the January 29, 2005 issue of Canadian TV Guide.

In 1998, he starred as Gary Lamb – Ground Activist in a series of Nike commercials directed by Smith. From 2010-2012, Mewes was cast as Jimmy the Janitor for 26 episodes of the Canadian television series Todd and the Book of Pure Evil.

In the video game Scarface: The World Is Yours, Mewes provides a voiceover for a thief who steals some tapes.

Mewes starred in the horror film Breath of Hate, where he played a romantic love interest to a dysfunctional escort. He also co-starred in Smith's 2008 film Zack and Miri Make a Porno.

Mewes co-hosts a weekly podcast with Smith called Jay & Silent Bob Get Old in which Mewes discusses his former drug addiction issues and is co-Executive Producer on Smith's Hulu-exclusive series Spoilers with Kevin Smith. He also co-produced the 2013 animated movie Jay & Silent Bob's Super Groovy Cartoon Movie with his wife Jordan Monsanto, which was based on the Bluntman and Chronic comic book that Kevin Smith wrote as a companion piece to the film Jay and Silent Bob Strike Back.