The actor has played anaesthetist Keith Greene on a recurring basis since 2005, clocking up 75 episodes on the show. It is thought that he will now concentrate on other projects.
Along with his Holby role, MacQueen is known for his comedy career, having appeared in sitcoms The Thick of It, Peep Show, The IT Crowd and The Inbetweeners in recent years.
He has also had film roles in 2005's Keeping Mum and 2007's Magicians.
In a recent interview with DS, Holby's series producer Diana Kyle had confirmed that more departures were on the way for the programme. She also promised that "several new cast members" will soon be introduced to the show.