Boris Johnson has urged Conservative MPs to back his plan to override part of the Brexit withdrawal agreement.
In a Zoom call with around 250 of them, he said the party must not return to "miserable squabbling" over Europe.
The EU has warned the UK it could face legal action if it does not ditch controversial elements of the Internal Market Bill by the end of the month.
And a Tory MP has proposed an amendment to the bill, which would affect trade between Britain and Northern Ireland.
Meanwhile, the European Parliament has threatened to scupper any UK-EU trade deal if the bill becomes UK law.
The two sides have less than five weeks to agree a deal before Mr Johnson's 15 October deadline - after which he says he is prepared to "walk away".
Informal talks are due to resume on Monday, with the next official round of talks - the ninth since March - starting in Brussels on 28 September.
The Internal Market Bill, which will be formally debated in the House of Commons for the first time on Monday, addresses the Northern Ireland Protocol - the part of the Brexit withdrawal agreement designed to prevent a hard border returning to the island of Ireland.
If it became law it would give UK ministers powers to modify or "disapply" rules relating to the movement of goods between Britain and Northern Ireland that will come into force from 1 January, if the UK and EU are unable to strike a trade deal.
The EU says the planned changes must be scrapped or they risk jeopardising the UK-EU trade talks.
But the government has rejected this demand, arguing the measures in the bill are needed to protect the integrity of the UK and the peace process in Northern Ireland.
In his Zoom call with MPs on Friday, the prime minister did not take questions and a poor signal meant the video and audio connections were lost for several minutes.
He called for "overwhelming support" for the bill, describing it as "absolutely vital" to "prevent a foreign or international body from having the power to break up our country".
Mr Johnson added that he would not countenance "the threat of a border down the Irish Sea".
But he said there was still a "very good chance" of the UK and EU striking a deal by mid-October similar to that previously agreed between the EU and Canada - which got rid of most, but not all, tariffs on goods.
BBC chief political correspondent Vicki Young said Tory MPs were "looking for a sign of compromise" from Mr Johnson, as they "simply can't believe the government is prepared to break international law", but the prime minister "dug his heels in".
'A harmful act'
In a column in the Daily Telegraph, Mr Johnson accused the EU of reinterpreting the Withdrawal Agreement to "destroy the economic and territorial integrity of the UK" and "endanger peace and stability in Northern Ireland".
"I have to say that we never seriously believed that the EU would be willing to use a treaty, negotiated in good faith, to blockade one part of the UK, to cut it off," he said.
Conservative backbencher Sir Bob Neill, who chairs the Commons Justice Committee, said he was not reassured by the prime minister's Zoom call.
He is tabling an amendment to the bill to try to force a separate parliamentary vote on any changes to the Withdrawal Agreement.
"I believe it is potentially a harmful act for this country, it would damage our reputation and I think it will make it harder to strike trade deals going forward," he said.
At around the same time as the prime minister was speaking, the European Parliament announced it would "under no circumstances ratify" any trade deal reached between the UK and EU if the "UK authorities breach or threaten to breach" the withdrawal agreement.
Northern Ireland Secretary Brandon Lewis has admitted parts of the bill, which would go against a treaty signed by the UK and EU, would "break international law in a very specific and limited way".
There is unease over this within the Conservative Party, with former leaders Theresa May, Lord Howard and Sir John Major urging Mr Johnson to think again.