The MPs voted through the Letwin amendment to delay Brexit until necessary UK legislation is passed, the BBC reported.
Johnson, who opened the debate soon after Parliament sat for discussion on his Brexit deal saying his deal with the EU can "heal the country", had to deal with disappointment.
The British Parliament sat for the first time in 37 years on Saturday to take up Johnson's Brexit deal agreed with the EU.
There were loud cheers at the People's Vote rally in Parliament Square as the Letwin amendment was passed in the House of Commons.
The latest development implies that Parliament will not vote on Johnson's deal until next week.
The division list for the Letwin amendment shows 231 Labour MPs voted for it alongside the 10 Democratic Unionist Party MPs.
They were joined by 19 Liberal Democrats, 35 Scottish National Party members, 17 Independents, four Plaid Cymru, five Independent Group for Change and the Green Party MP Caroline Lucas.
On the other hand, 283 Conservative MPs voted against the Letwin amendment along with six Labour MPs.
The six Labour MPs who rebelled to oppose the Letwin amendment were Kevin Barron (Rother Valley), Ronnie Campbell (Blyth Valley), Jim Fitzpatrick (Poplar and Limehouse), Caroline Flint (Don Valley), Kate Hoey (Vauxhall), and John Mann (Bassetlaw).
The Tory MPs left the Commons' chamber en masse, following the government's loss over the Letwin amendment. According to a BBC report, just a few of them were left.
Chief spokeswoman for the European Commission, Mina Andreeva, tweeting on the development said: "@EU_Commission takes note of the vote in the House of Commons today on the so-called #Letwin Amendment meaning that the #WithdrawalAgreement itself was not put to vote today. It will be for the UK government to inform us about the next steps as soon as possible."
Johnson, however, insisted that he will not negotiate a delay with the EU even as the move by the MPs has forced him to ask the EU for another Brexit delay. It's called the Benn Act, that comes into effect at 11 p.m. (local time) on Saturday night.
Johnson, however, said: "I will not negotiate a delay, nor does the law force me to."
Responding to this, the House of Commons in its Twitter account has clarified that the law mandates the UK PM to request a Brexit delay.
"The House of Commons has approved the amended motion on the new Brexit deal agreed between the UK Government and the EU," it said, adding: "The government must ask for an extension of Article 50 under the Benn Act and set out how it intends to proceed."
The Letwin amendment was proposed by Oliver Letwin, an MP who was booted by Johnson out of the Conservative parliamentary party in September after he supported an anti-no-deal legislation known as the Benn Act.
The amendment, supported by the MPs on Saturday, calls for the House to "withhold support" from the UK PM's Brexit deal with the EU until all legislations needed to implement the bill are passed by Parliament as well.
( With inputs from IANS )