Peers voted by 366 to 268 to amend the bill empowering May to trigger Article 50 of the EU's Lisbon Treaty, the formal notification of Brexit which she has promised to issue by the end of March.
She remains confident she will meet that deadline, but the defeat is a setback -- as well as a taster of the trouble she could face from lawmakers as she embarks on complex EU negotiations.
The amendment would give parliament the power to reject the final Brexit deal agreed with the EU -- a move critics said was akin to a "veto" which would damage the government's negotiating hand.
Ministers would still be constrained by the strict Article 50 timetable, however, which means that Britain will leave the EU after two years whether it has struck a withdrawal agreement or not.
The defeat comes after peers defied the prime minister for a first time on March 1, to include in the bill guarantees for more than three million European citizens living in Britain after Brexit.
The bill will now return to the elected House of Commons for debate, likely on March 13, where May's Conservatives have a majority and should be able to overturn both amendments.
With an eye on future parliamentary battles ahead, some senior Conservatives are urging a snap election to bolster May's support among MPs.
"The government could face many close votes, concessions or defeats as it tries to implement Brexit," former Tory leader William Hague wrote in the Daily Telegraph.
However, changing the law to bring the election forward from 2020 would not be easy, and for now Downing Street is publicly ruling it out.
Incentive for a bad deal
May had urged the Lords not to amend the two-clause bill, saying it was designed only to implement the June referendum vote for Brexit, and offered reassurances to address peers' concerns.
She promised to address the future of EU nationals in Britain early in the Brexit talks, and also insisted that parliament would get to vote on the final deal.
But this vote would only take place if May decides to accept the agreement on offer -- and she has warned she would rather walk away with nothing than accept a bad deal.
Opponents fear this would cause chaos, as all trade deals and contracts between Britain and its 27 former EU partners would become void overnight, and say parliament must make the final call.
Former Conservative minister Michael Heseltine backed the Labour amendment, warning of the huge uncertainty as Britain implemented the "most momentous peacetime decision of our time".
"I must make clear that in accepting the mandate to negotiate our withdrawal from the European Union, I do not accept that the mandate runs for all time and in all circumstances," he said.
But another Tory grandee, Nigel Lawson, said the amendment would allow parliament to "prevent Brexit altogether by refusing to allow the UK to leave the EU without agreement".
George Bridges, from the government's Brexit ministry, told the House of Lords: "By denying the prime minister's ability to walk away from the negotiating table, this would only incentivise the Euorpean Union to offer us a bad deal".
Last week's amendment on EU nationals dashed May's hopes of securing approval for the bill this week, ahead of an EU summit starting on Thursday.
She should easily be able to overturn it in the Commons, although reports suggest that on the second amendment on a final vote, she could face a rebellion from up to 20 of her Conservative MPs.
Among them is former minister Anna Soubry, who warned at the weekend: "If we are faced with a potentially catastrophic 'falling off a cliff', the least we can do is provide a parliamentary safety net."
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