MPs were set to begin debating amendments to the bill on Wednesday that could radically alter it or wreck its prospects of passing.
Among alternative plans being pushed by MPs are holding a second referendum and compelling Johnson to negotiate retaining ties to EU customs rules after Brexit.
Another amendment designed to extend the 14-month transition period beyond 2020 is also gathering support among MPs.
The rejection of Johnson’s timetable means the government will have to decide whether to bring forward a new timetable or attempt to force a general election.
The prime minister earlier on Tuesday insisted that any attempt to frustrate or delay Brexit would mean that he would pull the bill and “go forward with a general election,” adding that Brexit could not be allowed to continue to be delayed for months.
Sources close to Johnson suggested that he might be willing to accept a shorter, weeks-long delay to ratify his deal, according to multiple reports.
Donald Tusk, the European Council president, indicated on Tuesday that the EU was prepared to grant an extension of the Brexit deadline until next year.
Tusk told the European Parliament: “The situation is quite complex following events over the weekend in the UK, and the British request for an extension of the Article 50 process.”
He added: “We should be ready for every scenario. But one thing must be clear, as I said to Prime Minister Johnson on Saturday: A no-deal Brexit will never be our decision.”
Before the vote, the Labour Party leader, Jeremy Corbyn, said the prime minister’s deal was “nothing less than a charter for deregulation and a race to the bottom.”
“They always say the devil is in the detail,” he said. “And having seen the detail, it confirms everything we thought about this rotten deal: a charter for deregulation across the board, paving the way for Trump-style trade deals that will attack jobs, rights, and protections and open up our precious National Health Service and other public services for further privatisation.”
What’s in the Withdrawal Agreement Bill?
The Withdrawal Agreement Bill is designed to give legal effect to the Brexit deal negotiated by Johnson and allow Britain to legally leave the EU while entering a 14-month transition period.
The bill is based on a previous agreement negotiated by May but with significant differences in the relationship between Northern Ireland and the rest of the United Kingdom.
Under the agreement, Northern Ireland would remain tied to EU customs rules after Brexit. This measure has proved hugely controversial because it would mean new checks in the Irish Sea, something that has outraged unionists in Northern Ireland.
Other aspects of the deal include provisions for the UK to pay its £39 billion “divorce bill” to the EU and for maintaining the rights of EU citizens living in the UK after Brexit.
Opponents of the bill are concerned about several other aspects of the agreement, including provisions that would allow the prime minister to take Britain out of the EU without a deal at the end of the transition period.