The Commons voted by 315 votes to 274 for Labour MP Hilary Benn’s amendment. which effectively prevents the next Prime Minister from pushing through a no deal by preventing MPs from having a say.
Digital minister Margot James resigned after voting against the Government.
Chancellor Philip Hammond, Justice Secretary David Gauke, Business Secretary Greg Clark and International Development Secretary Rory Stewart defied orders and abstained from the vote.
Tory leadership contender Jeremy Hunt said he thought he was given permission to miss the vote, but was mistaken.
He tweeted: “I missed votes today because I thought I was slipped and it turns out I was not. Apologies to my colleagues & Whip. My position is that parliament should NOT restrict the hands of an incoming govt in this way & I remain opposed to how parl voted.”
His rival Boris Johnson voted against the move to block prorogation.
Theresa May said she was “disappointed” that multiple ministers failed to vote on the amendment - but failed to take action against them.
However, she issued a warning to the MPs over whether their roles will still be available under her successor.
A spokesman said: “The Prime Minister is obviously disappointed that a number of ministers failed to vote in this afternoon’s division.
“No doubt her successor will take this into account when forming their government.”
Opposition and Remain MPs were jubilant, with Shadow Brexit Secretary Keir Starmer claiming a ‘huge victory’.
Justice Secretary David Gauke was rumoured to be considering resigning after he said proroguing Parliament would be "outrageous".
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He told BBC Radio 4's Today programme earlier this morning: "I will have to see what the precise amendments are and we're hearing what the whipping will be and the arguments for that so I'm not in a position to necessarily say.
"But what I would say is the idea that Parliament should be suspended in October - a period where it always sits, Parliament has always in recent years sat at that time of year.
"And at a crucial point in this country's history, if you like - that Parliament should not be able to sit, should not be able to express its opinion and its will, I think would be outrageous.”
Front runner for the Tory leadership Mr Johnson again refused at the final campaign hustings on Wednesday to rule out proroguing - suspending - Parliament in order to meet his red line of getting the UK out of the EU by October 31.
Mr Johnson’s team are rumoured to have devised a plan to hold the Queen’s Speech, in which the PM lays out his policy programme, in November.
Such a move would mean MPs would be sent home two weeks before it - effectively suspending Parliament and allowing a no-deal Brexit to happen without any interference.
Rival for Number 10 Jeremy Hunt has insisted he would not use such a constitutional manoeuvre to force EU withdrawal.