After losing a safe Tory seat and facing a third of his MPs revolting last week, the PM has now lost a critical supporter.
Lord Frost, the Brexit negotiator, resigned immediately after the Mail on Sunday revealed the news.
He wanted to leave last week, but Mr Johnson persuaded him to stay until January.
‘It is sad that this proposal has become public this evening and in the circumstances I believe it is right for me to write to step down with immediate effect,' Lord Frost wrote to the PM on Saturday evening.
Lord Frost congratulated Mr Johnson and stated that ‘Brexit is now secure', but added that ‘the job for the Government now is to deliver on the opportunity it affords us'.
‘You know my worries about the current course.'
'I hope we can get back on track soon and not be lured by the kind of forceful actions we have seen elsewhere,' he added.
Lord Frost reportedly objected to the introduction of Plan B measures, such as Covid passes for large stadiums.
The cost of net zero climate change promises and tax increases further disillusioned him.
Lord Frost has recently been in difficult talks with the EU regarding the Northern Ireland Protocol.
Britain claims the EU applies the convention too strictly and wants to abolish customs checks between the UK and Northern Ireland.
Lord Frost stated on Friday that he was disappointed that he couldn't reach a new deal before the end of the year.
After speaking with Vice-President Maros Sefcovic on Friday, he suggested talks would resume in 2022.
It was not as much or as swiftly as we had hoped for, he said.
Labour's deputy leader Angela Rayner said the revelation showed a "government in absolute turmoil" at a time of uncertainty.
‘@BorisJohnson isn't up to it. We deserve better.'
On Thursday, days after 100 of his MPs voted against his new Covid plans, Mr Johnson's Conservatives lost a by-election in North Shropshire to the Liberal Democrats.
Andrew Bridgen, a Conservative backbencher, told Mr Johnson that he was ‘running out of time and friends' to deliver on Conservative commitments and discipline.
Lord Frost, 100 Conservative backbenchers, and most importantly, the people of North Shropshire have made their views known.
Northern Ireland politicians had hoped Lord Frost might rewrite the dreaded protocol.
Lord Donaldson said Mr Johnson's resignation was a terrible indication for his commitment to abolishing the Irish Sea border.
‘This government is sidetracked by internal bickering,' Sir Jeffrey remarked.
Noel: But this raises fundamental problems for the Prime Minister and his approach to the NI Protocol.'