The House Of Cards actor stars in legal drama Clarence Darrow to celebrate the end of his 10 years as artistic director of The Old Vic theatre in London.
In character as the 19th-century America lawyer, Spacey relives some of the experiences that established his reputation as a courtroom giant and civil rights hero.
During a scene in which his character is on trial, Spacey was addressing the audience with a passionate plea for his innocence, when a mobile phone began ringing in that section.
The music got louder and louder as the guilty party tried to look innocent, until Spacey finally snapped, in character: "If you don't answer that, I will!" He received a round of applause.
He is not the first actor to rebuke an audience member.
Last year Hollywood actor James McAvoy stopped a performance of Macbeth he was appearing in at Trafalgar Studios in London when he saw a man filming him. He refused to continue until he had put the device away.
The late Richard Griffiths also tore into an audience member while performing in The History Boys after a mobile phone began ringing, and the Scottish actor Ken Stott, when appearing in A View from the Bridge in 2009, stopped the play because of some noisy schoolchildren.