"This deal you can't do it, you can't trade. We can't make a trade deal with the UK," he said in an interview with Brexit campaign figurehead Nigel Farage on the UK's LBC radio.
Trump's comments stand at odds with his previous pledge in September that he was working closely with Johnson to strike a "magnificent trade deal" once Britain left the European Union.
Johnson struck the terms for the UK's departure with Brussels earlier this month but has been unable to push it through parliament. He hopes a general election, scheduled for December 12, will break the deadlock, giving him a majority that will help him get the enabling legislation approved.
Boris Johnson named new British prime minister
The US president's intervention came effectively on the first full day of campaigning that saw Johnson tour a school and don a white coat for a hospital visit.
In parliament, on Wednesday he said he had struck a "fantastic deal" with the EU, and on Thursday promised Britain would meet the latest Brexit deadline of January 31 next year.
Trump told Farage "we can do many times the numbers that we're doing right now - and certainly much bigger numbers than you're doing under the European Union".
Farage, a close Trump ally, criticised then-US president Barack Obama in 2016 for "interfering" in British democracy after he commented on possible US-UK trade deals post-Brexit.
The former City trader has remained tight-lipped about speculation he could form an electoral pact with Johnson to avoid splitting the pro-Brexit vote.
Farage's Brexit Party, which won the biggest share of votes at European Parliament elections in May, has its official campaignlaunch on Friday.
Johnson seeks to push Trump at fractious G7
But Trump said: "I'd like to see you and Boris get together because you'd really have some numbers, because you did fantastically in the last election, and he respects you a lot."
He added: "I think it'd be a great thing."
Farage said he would be "right behind" Johnson but only "if he drops this dreadful deal".
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