Hecklers stood up and chanted repeatedly "we believe the women" at Bill Cosby's third and final performance of a Canadian tour.
The hecklers then marched out of the theatre in Hamilton, Ontario, as the 77-year-old comedian asked his audience not to react.
Cosby is facing growing sexual assault allegations from more than 15 women, with some claims dating back decades. He has denied the allegations through his lawyer and has never been charged.
The walk-out during last night's show came a day after a man at Cosby's performance in London, Ontario, called the comedian "a rapist".
It also took place just minutes after Cosby acknowledged the controversy that has followed him throughout his three-city Canadian tour.
"Whatever happens here tonight if there's some sort of outburst, we just have to remain calm and things will be taken care of. It oughtn't last that long," he said.
"I want to thank all of you here tonight because you stayed with what you believed in."
An announcement at the start of the show warned the audience there might be attempts to disrupt the show and urged ticket holders to remain calm until the matter was resolved.
Security at the venue was tight - bags were searched and police officers and private security guards were posted along the aisles of the theatre.
The three shows in Canada were Cosby's first since November when he had a string of performances cancelled over the accusations.