Under the terms of the amendment, the powers in the bill can only come into force if MPs have approved a Brexit deal, Article 50 has been extended, or MPs have given their backing for leaving the European Union without a withdrawal agreement.
The defeat highlights Mrs May's weak position as leader of a minority government, a divided party, and a critical Parliament just days before she is due to hold a pivotal vote on whether to approve the Brexit deal she has negotiated with the EU.
The Commons amendment, tabled by Labour's Yvette Cooper, is created to limit the Treasury's ability to spend money on no-deal preparations, without the explicit consent of Parliament. That trial of strength is a sign of the running battles that the Commons will face over the next week as the Brexit debate reaches its climax.
Downing Street described it as an "inconvenience" which would not prevent the Government collecting taxes. Critics of May's deal to smooth Britain's passage out of the European Union on 29 March say the backstop could leave Britain subject to European Union rules indefinitely, long after it gives up any say over them.
Meanwhile, a European Commission spokesman played down talk of a possible renegotiation of the Brexit deal which has been agreed, saying, "Everything on the table has been approved and the priority now is to await events in the United Kingdom".More news: Justin Fields pens note to Georgia fans on Twitter
Ireland's economy would be hit hard by a disorderly Brexit, and the most contentious part of May's deal is an insurance plan aimed at preventing a hard border between the Irish Republic and Northern Ireland.
However, Treasury minister Robert Jenrick told Parliament the proposal's only impact would be to make the country "somewhat less prepared" for a crash out of the bloc.
As Sky's political editor Faisal Islam tweeted, "this is the start of constitutional trench warfare where Parliament will try to assert its power over the executive to prevent No Deal - highly significant".
The Labour leader said ministers were "fooling nobody" with preparations for no-deal, and said Mrs May would not be allowed to run down the clock to secure support for her Brexit deal.More news: How did refs make this impossibly bad Kevin Durant call?
Environment Secretary Michael Gove told cabinet colleagues that critics holding out for a better deal were like swingers in their mid-50s waiting for Hollywood star Scarlett Johansson to turn up on a date.
Work and Pensions Secretary Amber Rudd said: "History will take a dim view of a cabinet that presses ahead with no deal".
With parliament deadlocked, the ultimate destination of the Brexit project remains unclear.
"The majority tonight that is expressed in this house will sustain itself". Anything will need legislation.
A source in Brussels told AFP several weeks ago that Britain has been discussing the possibility with European officials, and a junior British minister, Margot James, publicly voiced the idea on Monday. "Ensuring that we actually get the future relationship in place to replace the backstop if it's used is actually a crucial element of this".More news: Windows 10 edges out Windows 7 as most popular operating system
"I'm anxious we could come to the crunch and parliament will not have the powers to stop [no deal] happening" Cooper said in the aftermath of the vote, adding: "I think we have a responsibility not to just stand by".