Pemberton attended St. Michael's CE High School in Chorley, Lancashire where his acting skills were first realised. Pemberton graduated from Bretton Hall College with a BA (hons) in Theatre Arts.

His previous work has centred mainly around fringe theatre, being a founder member of 606 Theatre with Gordon Anderson, Tom Hadley and producer Shane Walter, and he has produced, performed in and directed stage productions. Pemberton has written for Variety and was the assistant editor of the International Film Guide from 1991 to 1998.

He is best known as a member of the sketch comedy team The League of Gentlemen, along with fellow performers Mark Gatiss, Reece Shearsmith, and co-writer Jeremy Dyson, all of whom he met at Bretton Hall drama school in his late teens. The League of Gentlemen initially began as a stage act in 1995, transferred to BBC Radio 4 as On the Town with the League of Gentlemen in 1997 and then arrived on television on BBC Two in 1999. The latter has seen Pemberton and his colleagues awarded a British Academy Television Award, a Royal Television Society Award and the prestigious Golden Rose of Montreux.

Pemberton's other TV credits include: Whitechapel (2009); Doctor Who (2008); Benidorm (2007-present); Under the Greenwood Tree (2005); Hotel Babylon (2005); The Last Detective (2005); Randall & Hopkirk ; and Blackpool. He also appeared in Shameless as the husband of Sheila, Frank's Girlfriend. In 2004 he played Dr. Bessner in Agatha Christie's Poirot — Death on the Nile. Pemberton also starred in the film Lassie (2005) as Hymes.

2007 saw him make an appearance in the official Comic Relief film Mr Bean's Holiday as the vicar in the first scene.

In July 2007, Pemberton took over from Bob Martin as "Man in Chair" in the West End production of the musical The Drowsy Chaperone, playing the role until the production closed on August 4, 2007.

In the 2008 English language DVD re-release of the cult 2006 Norwegian animated film Free Jimmy, Pemberton voiced the character of "Mattis", a heavy-set, bizarrely dressed biker member of the "Lappish Mafia". In this his voice is used along with the other actors of League of Gentlemen such as Reece Shearsmith and Mark Gatiss. The dialogue was written by Simon Pegg and other actors, with performances by Pegg himself, Woody Harrelson, and others.

More recently in 2009 he has written and starred in Psychoville alongside Reece Shearsmith from The League of Gentleman once more.