Gillen was born in the Drumcondra suburb of Dublin, Ireland. He began his acting career as a teenager with Dublin Youth Theatre, at the age of 16 playing Bottom in A Midsummer Night's Dream at the Project Arts Centre, before moving to London.

He is perhaps best known in the UK for his role as Stuart Alan Jones in the ground-breaking British television series, Queer as Folk, and its sequel, Queer as Folk 2, for which he was nominated for the British Academy Television Award for Best Actor in 2000.

American audiences know him best for his role as Tommy Carcetti in HBO's television series The Wire.

Gillen was nominated for a Tony Award for his highly acclaimed Broadway role in Harold Pinter's The Caretaker in 2003. He has also been nominated for an Irish Times Theatre Award for his portrayal of Teach in the Dublin Gate Theatre's 2007 production of David Mamet's American Buffalo, and in its London West End production.