Mat Horne studied drama at the University of Manchester, where he met Bruce Mackinnon, and formed a comedy double act, making the final of the So You Think You're Funny new act competition at the 2000 Edinburgh Festival.

His first TV appearance was in the obscure E4 show Noble and Silver: Get Off Me! in 2001, and he had a number of small roles until landing the part of RE teacher Ben Birkett in Channel 4's Teachers in 2004. He was a regular in the third series of The Catherine Tate Show in 2006, and starred in the BBC sitcom Roman's Empire the following year.

However his big break came with BBC Three hit Gavin & Stacey in 2007, which earned him a best male newcomer nomination at that year's British Comedy Awards. On the show, he struck up a friendship with co-star (and co-writer of the series) James Corden. They have since hosted Big Brother's Big Mouth together, as well as various awards ceremonies. Latterly they have been working together on a BBC sketch show, and a movie Lesbian Vampire Killers.