"Let us end the process of having people with bigoted religious views treating women as second class citizens," he told Labour MPs
"I think you should all take the pledge - no more segregated meetings," he added.
It follows reports of Labour meetings where Asian men and women have been separated along gender lines.
A Labour Party spokesperson said: "David Cameron is trying to imply that the Labour Party condones segregation.
"This is absolutely untrue. We make sure that everyone is treated equally and respectfully and our record on gender equality speaks for itself."
In May last year, Labour's then deputy leader Harriet Harman told LBC radio she did not "agree" with a Labour rally she had attended in Birmingham where Asian men and women were seated on different sides of the room.
She said it would have better if the meeting had decided to "sit round in a big circle" but she did not "like to be rude" by walking out.
"This meeting was organised by a male Labour councillor and a female Labour councillor, and what they were trying to do is ensure there weren't men-only meetings, which we are even more not in favour of," she told LBC.
"It was actually bringing the women from the community into the meeting."
At the time the Labour Party said: "There was no forced segregation. Speakers at the event included both women and men."
Labour's current deputy leader Tom Watson, Ms Harman's husband, Labour MP Jack Dromey, and Birmingham MPs Liam Byrne and Khalid Mahmood were also reported to be at the meeting.
David Cameron has called on Labour to end "segregated" party meetings, at Prime Minister's Questions.