Jimmy Akingbola (Antoine Malick), Lauren Drummond (Chantelle Lane) and Ty Glaser (Gemma Wilde) will all bow out on screen later this year.
Akingbola, who joined the show in January 2011, commented: "I feel honoured to have been given the opportunity to play such an iconic and groundbreaking character like 'The Malick' in Holby City. It has been a dream come true.
"We created a special, unpredictable individual who has been successfully embraced by the viewers and which has brought a whole new audience to Holby City. Holby is and will always be my family - the cast, crew and production team are all fantastic and I will miss them all."
On leaving the role of Chantelle, Drummond added: "This job has exceeded all my expectations and it has been an absolute joy to be a part of creatively and personally. The cast and crew are one of a kind!"
Meanwhile, Glaser said of her upcoming exit: "I've had such a wonderful time working on Holby - the cast and crew have all been exceptional, a joy to work with. I'm beyond excited about future projects ahead of me but will always remember Holby as a fantastic year in my career."
News of the cast departures follows the recent announcement that Guy Henry has filmed his final scenes as Henrik Hanssen. All four characters have been tipped to have "nail-biting and life-changing individual storylines" in the next few months.
Holby City's executive producer Oliver Kent said: "Each of these unforgettable characters has brought something unique to Holby City and it has been an absolute pleasure having them on the show.
"I'd like to thank Guy, Lauren, Jimmy and Ty for putting in stellar performances week in and week out to bring these characters to life and I wish them all the best for the future."
With a handful of cast members leaving, show chiefs have already signed up new faces to shake up life at the hospital. Coronation Street actor John Michie has landed the part of consultant neurosurgeon Guy Self, while Law and Order: UK actress Camilla Arfwedson has joined as plain-speaking junior doctor Zosia March.
No Angels star Louise Delamere has also now been added to the cast, playing Holby's new director of nursing Colette Sheward.
Delamere said: "It's great to be joining Holby City as part of the team. Colette is a surprising and funny character and I look forward to starting my ward rounds!"
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