The prime minister now faces the prospect of court action after telling MPs he will not negotiate an extension to the Article 50 Brexit talks beyond 31 October as required by the law.
And speaker John Bercow hinted he may strike down a desperate attempt by the government to re-run on Monday the “meaningful vote” on Johnson’s Brexit plan that was thwarted by MPs amid dramatic scenes in the first Saturday sitting of parliament since the Falklands War.
The PM’s defeat by a margin of 322 votes to 306 was greeted by a roar of delight from thousands of demonstrators gathered outside parliament for the final rally of the Together for the Final Say march, staged by People’s Vote and backed by The Independent.
They heard speakers from across the political spectrum, including an array of senior Labour figures including shadow chancellor John McDonnell, insist that any Brexit deal must be subject to a confirmatory referendum.
Declaring that Labour – despite the absence of its leader Jeremy Corbyn – is a Remain party, Mr McDonnell said: “We believe that our future best lies within the European Union itself.”
SNP MP Joanna Cherry vowed to ask Scotland’s highest court on Monday to take action to force Johnson to request a delay to Brexit.
And Mr Bercow said he was ready to sign the letter to Brussels in Johnson’s place if asked to by Edinburgh’s Court of Session, telling Ms Cherry: “If I were instructed by this House, I would do as instructed and if I were directed or instructed by a court, I would do as directed.”
Johnson struck a defiant tone after seeing his hopes dashed of finally securing parliamentary approval for an EU withdrawal deal, after three failed attempts by his predecessor Theresa May.
His plan was scuppered by an amendment tabled by former Conservative cabinet minister Oliver Letwin, who he last month expelled from the party over a previous Brexit rebellion.
Despite saying he wanted to see Mr Johnson’s deal succeed, Letwin pushed through a provision withholding MPs’ approval until all stages of legislation to ratify it have been completed.
The move was a remarkable demonstration of the lack of trust in the prime minister from Tory exiles, who feared that victory for the PM would free him to crash the UK out of the EU without a deal in 12 days’ time by failing to legislate for the agreement’s implementation.
Instead of confirming he would comply with the law requiring him to delay Brexit, Johnson took to the despatch box to declare: “I will not negotiate a delay with the EU, neither does the law compel me to do so.”
Mr Corbyn insisted the PM must obey the law, telling him: “He can no longer use the threat of a no-deal crash-out to blackmail members to support his sell-out deal.”
The European Commission said it “took note” of MPs’ vote and expected the UK government to “inform us about the next steps as soon as possible”. In a statement, Irish premier Leo Varadkar noted that an extension beyond 31 October “can only be granted by unanimity” of the 27 remaining EU leaders.
Commons leader Jacob Rees-Mogg said the government was planning to give MPs another chance on Monday to approve the last-minute deal sealed by Johnson in Brussels on Thursday.
But Mr Bercow signalled he may not allow the motion to be put forward again so soon, telling MPs it would be “most curious and irregular” if its purpose was to seek to invalidate today’s vote.
And a Tory rebel source accused the PM of “petulance”, pointing out that the successful Letwin amendment states that approval cannot be granted “unless and until” ratification through a Withdrawal Agreement Bill is complete.
“This sends a clear signal that the prime minister should now bring forward the WAB, not a meaningful vote, on Monday so that MPs can vote for or against it,” said the source. “In the meantime, he must comply with the law.”
Mr Johnson insisted he was not “dismayed or daunted” by the setback.
If he is unable to secure a meaningful vote on Monday, he will hope to reverse his defeat during the passage of the Withdrawal Agreement Bill over the coming days.
But scrutiny of the bill is likely to be a fiercely-fought affair, giving MPs plenty of opportunities to insert amendments to require a second referendum.
People’s Vote organisers estimated the size of the demonstration at up to 1 million, making it one of hte biggest public protests in British history. Marchers and supporters flocked to sign a letter to MPs, MEPs, the prime minister and the leaders of the other 27 EU states calling for a Final Say ballot.
Ministers including Mr Johnson’s no-deal supremo Michael Gove were given police escorts to leave a Palace of Westminster surrounded by thousands of protesters, leading business secretary Andrea Leadsom to complain of “intimidation”.
The Final Say letter can be signed at www.peoples-vote.uk/letter.