May vowed to fight for the leadership of her party and the country "with everything I've got", and spent the day holed up in the House of Commons trying to win over enough lawmakers to secure victory.
One member of the government told me the result of the ballot would be closer than expected - not a comfortable victory.
The leadership challenge marks a violent eruption of the Conservative Party's decades-long divide over Europe.
Environment secretary Michael Gove added, "I think the PM will win tonight and she will win handsomely".
Pro-Brexit lawmakers Jacob Rees-Mogg and Steve Baker said in a joint statement that "in the national interest, she must go".
A claim of reaching the threshold turned out to be premature before, but anger over May's handling of Brexit has been growing within her party.
Less than four months before the United Kingdom is due to leave on March 29, Brexit is in chaos, with options ranging from a potentially disorderly no-deal departure to another referendum that could reverse the whole thing.
British Prime Minister Theresa May's job is on the line in a non-confidence vote by Conservative MPs. If there is no satisfactory deal by then, parliament will still be given a debate on the issue.
"She's not the person to see Brexit through", he said.
But she could choose to quit immediately instead, with an interim leader taking over until the permanent replacement is found.
British business figures expressed alarm at the prospect of even more political uncertainty.
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Adam Marshall, director general of the British Chambers of Commerce, said politicians should understand the impact of their "high stakes gambles" with Britain's economy. That's the 15 per cent of Conservative legislators needed to spark a leadership challenge under party rules.
If she loses, a leadership election will then take place with a new Prime Minister likely to take office by mid-January. The victor of the vote becomes Conservative leader and prime minister, without the need for a national election.
If May wins, she remains in office and can not be challenged again for 12 months.
The delay to the so-called "meaningful vote" by MPs means that ratification may not be possible until as late as 10 weeks before the scheduled date of Brexit on March 29.
May needs a simple majority - from 159 of 317 Conservative lawmakers - to remain leader. But our party will rightly not tolerate it.
But before it, Cabinet colleagues rallied to May's support.
May won support from senior allies in her government, including potential leadership rivals such as Foreign Secretary Jeremy Hunt and Pensions Secretary Amber Rudd.
"We are just at the start of the negotiations and the start of the discussions", she said after meeting with the EU Council's Tusk.
Opponents say the backstop would bind Britain to the EU in an acceptable way and unable to leave the customs union unilaterally.
"There is no room whatsoever for renegotiation", Juncker told European Union lawmakers in Strasbourg, France, as he briefed them on the summit.
With less than four months left until the United Kingdom is due to leave the European Union on March 29, May's Brexit deal is floundering, opening up prospects that run the gamut from a disorderly no-deal divorce to calling Brexit off.