Gatiss's earliest professional writing work was in the form of several novels in Virgin Publishing's New Adventures series of Doctor Who stories, beginning with Nightshade in 1992. Gatiss is a long-time fan of the British television science-fiction series, preferring its style from the 1970s. Much of his writing has been devoted to the series, including the BBV video spin-off series P.R.O.B.E., four novels, two audio plays for BBV and two audio plays for Big Finish Productions.

He is best known as a member of the sketch comedy team The League of Gentlemen (along with fellow performers Reece Shearsmith, Steve Pemberton and co-writer Jeremy Dyson), which 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 Gatiss and his colleagues awarded a British Academy Television Award, a Royal Television Society Award and the prestigious Golden Rose of Montreux.

He met his League of Gentlemen co-writers and performers at Bretton Hall drama school in his late teens, which he began attending after finishing school and having spent a gap year travelling around Europe.

Outside of the League, Gatiss' television work has included writing for the 2001 revival of comic telefantasy Randall & Hopkirk (Deceased) and script editing the popular sketch show Little Britain in 2003, making guest appearances in both. In 2001 he guested in Spaced as a villainous government employee modelled on Agent Smith. In the same year he appeared in several editions of the documentary series, "SF:UK". Other acting appearances include the comedy-drama In the Red (BBC Two, 1998), the macabre sitcom Nighty Night (BBC Three, 2003), Agatha Christie's Marple as Ronald Hawes in The Murder at the Vicarage, 2004–2005 and the live 2005 remake of the classic sci-fi serial The Quatermass Experiment. A second series of Nighty Night and the new comedy-drama Funland, the latter co-written by his League cohort Jeremy Dyson, both featured Gatiss and aired on BBC Three in the autumn of 2005. He appeared as Johnnie Cradock, alongside Nighty Night star Julia Davis as Fanny Cradock, in Fear of Fanny on BBC Four in October 2006, and featured as Ratty in a new production of The Wind in the Willows shown on BBC One on 1 January 2007. He wrote and starred in the BBC Four docudrama The Worst Journey in the World, based on the memoir by polar explorer Apsley Cherry-Garrard.

He also took an on-screen role in one episode of Doctor Who in 2007 (Professor Lazarus in "The Lazarus Experiment"), making him only the third person — after Glyn Jones and Victor Pemberton — and the first of the new series to both write for and act in the programme. Also in 2007, he appeared as Robert Louis Stevenson in Jekyll, a BBC One serial by his fellow Doctor Who scriptwriter Steven Moffat. In 2008 he appeared in Clone as Colonel Black. He also made a guest appearance in Pemberton and Shearsmith's comedy series Psychoville. In 2010 he portrayed Malcolm Mclaren in the BBC drama Worried About the Boy which focused on the life and career of Boy George, and also appeared as Mycroft Holmes in the BBC drama Sherlock, which he co-created with Steven Moffat.

Gatiss appears frequently in BBC Radio productions, including the sci-fi comedy Nebulous and The Further Adventures of Sherlock Holmes story The Shameful Betrayal of Miss Emily Smith. In 2009 he was The Man in Black when BBC Radio 7 revived the character (originally played by Valentine Dyall and Edward de Souza) to introduce a series of five creepy audio dramas. He is also involved with theatre, having penned the play The Teen People in the early 1990s, and appeared in a successful run of the play 'Art' in 2003 at the Whitehall Theatre in London. In film, he has starred in Sex Lives of the Potato Men (2004) and had minor roles in Birthday Girl (2001), Bright Young Things (2003), Match Point (2005) and Starter for 10 (2006). The League of Gentlemen's Apocalypse, a film based on the television series, co-written by and starring Gatiss, was released in June 2005. He also plays the recurring character of Gold in the audio revivial of Sapphire and Steel produced by Big Finish Productions.

In the 2008 English language DVD re-release of the cult 2006 Norwegian animated film Free Jimmy, Gatiss voiced the character of "Jakki", 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 Steve Pemberton and Reece Shearsmith. The dialogue was written by Simon Pegg and other actors included Pegg himself and Woody Harrelson, amongst others.

Mark appeared in the stage adaptation of Pedro Almodovar's All About My Mother at the Old Vic in London from 25 Aug-24 Nov 2007. He won much critical acclaim for his portrayal of the semi-transsexual Agrado.

Mark was scheduled to perform in Darker Shores by Michael Punter, a ghost story for all the family, at Hampstead Theatre 3 December 2009 - 16 January 2010 but had to withdraw after a serious family illness. Tom Goodman-Hill took over his role.

In March 2010 he was a guest on Private Passions, the biographical music discussion programme on BBC Radio 3.