Members of parliament on Tuesday voted 432 to 202 to rebuff the deal, giving May a crushing defeat with a 230-margin.
“The House has spoken, and the government will listen,” May said following the vote, even as she predicted “more uncertainty, more bitterness and more rancor.”
The vote plunges the already divided country deeper into turmoil as it tries to solve several key issues in the Brexit process.
Opposition Labour Party leader Jeremy Corbyn called it “the greatest defeat” for a British government since the 1920s.
Following the vote, Labour tabled a no-confidence motion in May’s minority government which will be debated on Wednesday.
“She cannot seriously believe that after two years of failure that she’s capable of negotiating a good deal for this country,” Corbyn said.
But May said she still wants to fulfil her duty to deliver on Britain’s 2016 vote for leaving the EU.
If she wins Wednesday’s confidence vote she will meet the leaders of the opposition parties in a “constructive spirit” to discuss the way forward, May said.
“Given the urgent need to make progress, we must focus on ideas that are genuinely negotiable and have sufficient support in this House,” she added.
In her final plea to the parliament, May had said that a vote against the deal “is nothing more than uncertainty, division and the very real risk of ‘No Deal’ or ‘No Brexit’ at all.”
“We can choose unity over division,” May said before the vote. “I believe that we have a duty to deliver on that referendum vote. And to do so in a way that protects people’s jobs, and protects our security, and protects our union.”
Al Jazeera’s Paul Brennan, reporting from London, said Brexit was “raising a question over the whole principle of parliamentary democracy” in Britain.
“It’s pitting the government against parliament, and parliament against the people. What was initially a binary in-out question has now given rise to a whole kaleidoscope of complex options and uncertain outcomes,” he added.
The country could now face a so-called “no-deal” Brexit that critics claim will be economically disastrous, another referendum, either on Brexit terms or whether to leave the EU at all.
May, who now has three days to bring a revised plan back to parliament, will now most likely seek concessions from the EU, then put her deal to parliament a second time.
However, the EU has said it will not negotiate the deal again.
With the margin of her defeat, it is is more unlikely that EU would give May more concessions, Al Jazeera’s David Chater, reporting from Brussels, said.
“There may be little Theresa May can discuss with the European Commission President Jean-Claude Juncker.”
In a statement following May’s defeat, Juncker said he “regretted” the result of the vote.
“The risk of disorderly withdrawal of the United Kingdom has increased with this evening’s vote,” Juncker said.
Tuesday’s vote was initially scheduled to be held on December 11 but was postponed by May when it became clear she faced certain defeat.
Last week, May warned British legislators that if the plan was rejected, a catastrophe would follow.
“Doing so would be a catastrophic and unforgivable breach of trust in our democracy,” she wrote.
The UK is poised to leave the EU on March 29, two years after it triggered Article 50, the exit clause in the EU’s constitution, and kick-started arduous negotiations with European leaders over a divorce deal.
However, since reaching a deal in November, the agreement has come under fire from across the political spectrum, with opponents of the EU seeking a cleaner break and pro-European legislators pressing for a second vote on membership in the bloc.
A second referendum, however, has been opposed by both May and main opposition leader Corbyn.
What next for Brexit?
Here are the three main scenarios facing Britain while the clock ticks down to March 29, 2019 – the day it is scheduled to depart the EU after 46 years:
Try again: The British government and EU leaders say their agreement is the best compromise available, and despite her landmark defeat, May said on Tuesday it remained the only option.
Members of her Conservative party say the deal keeps Britain too close to the EU, while opposition parties say fails to protect economic ties with the bloc.
Both sides also hate a plan to keep open the Irish border, the so-called backstop, which could see Britain indefinitely follow European rules on trade.
There is nothing to stop the government bringing back the same deal again and again to the House of Commons until either MPs accept it, or seek to remove May, who faces a no-confidence vote on Wednesday called by the opposition Labour Party.
No deal: This is billed as the doomsday scenario that threatens to trigger a recession in Britain and markedly slow the EU’s economic growth.
It is the default option if the British parliament votes against the deal and there are no other solutions before March 29.
May’s agreement was meant to keep trade rules between the world’s fifth-biggest economy and its largest export market almost unchanged for a transition period running to the end of 2020.
Second referendum: EU supporters have been calling for another vote ever since the “Leave” campaign won the 2016 referendum, and demands have stepped up in recent months.
There is no law keeping Britain from doing it all over again, but many question whether this would be democratic.
It also threatens to be just as divisive, with opinion polls showing the country is still split over the issue.
May has warned another vote “would do irreparable damage to the integrity of our politics.”