The amendment, put forward by Labour's Hilary Benn, was passed with a majority of 41, with 315 MPs in favour. 274 MPs voted against the amendment.
Had it passed, it would have meant Parliament could be shut down.
The votes came after the Lords strongly backed a bid to block Parliament being suspended in order to facilitate a no-deal exit by a margin of 103 votes on Wednesday.
The change to the Northern Ireland (Executive Formation) Bill will require progress reports on restoring devolved government in Northern Ireland to be debated regularly in Parliament, effectively preventing it being prorogued.
Justice Secretary David Gauke said before the vote that proroguing Parliament would be "outrageous" - but he did not confirm whether he would back the measures to block it.
MPs react to the Government defeat
Theresa May said she is "disappointed" multiple ministers failed to vote on the amendment.
She also issued a warning to the MPs over whether their roles will still be available under her successor.
"The Prime Minister is obviously disappointed that a number of ministers failed to vote in this afternoon's division," a spokesman said.
"No doubt her successor will take this into account when forming their government."
Digital minister Margot James was among those voting against the Government. She later resigned her Government position, Downing Street said.
Labour MP Hilary Benn, who brought forward today's amendment to stop parliament being suspended, told ITV News: "It's shown by a significant majority, 41 votes, that the House of Commons has decided if, for any reason we had been prorouged, then the amendment we passed means that parliament would have to come back.
"So this is really an expression of view by the House of Commons by a large margin, that you're not going to get away with prorogation to try and deny the House of Commons."
He added: "And it's a message to the new prime minister, whoever is elected next week."
Former Conservative frontbencher Justine Greening, who voted for Hilary Benn's amendment, said: "I think it does show there is a level of disquiet at how Brexit is being handled and that there's a real need for the incoming prime minister to listen to the broad base of Conservative MPs in parliament."
Conservative MP John Whittingdale told ITV News: "I think the chances of the next prime minister trying to get no deal through by prorouging parliament are very slim.
"Where I think Boris Johnson and Jeremy Hunt ride is that no deal needs to be on the table to give credibility to our negotiating position with the European Union."
Independent MP Nick Boles tweeted he was "proud" to accompany the minister and Conservative MPs Steve Brine, Richard Harrington and Alistair Burt "through the Aye lobby".
"Heroes all of them," he said.
He later thanked Philip Hammond, Greg Clark, David Gauke and Rory Stewart for helping to back the amendment.
Shadow Brexit Secretary Keir Starmer tweeted: "For Boris Johnson to try to shut down Parliament to force through a destructive 'no deal' Brexit would be a constitutional outrage. Now it would also be unlawful. A huge victory."
Tory leadership front runner Johnson again refused at the final campaign hustings on Wednesday to rule out proroguing Parliament in order to meet his red line of getting the UK out of the EU by October 31.
His rival for Number 10 Jeremy Hunt has insisted he would not use such a constitutional manoeuvre.
A source close to leadership candidate Mr Hunt said he was given permission to not take part in the vote.
Contrary to what his camp said earlier, Mr Hunt later confirmed he was not given permission to miss the vote.
He wrote on Twitter: "I missed votes today because I thought I was slipped and it turns out I was not.
"Apologies to my colleagues and whips office.
"My position is that parliament should not restrict the hands of an incoming government in this way and I remain opposed to how Parliament voted."