Commons leader Andrea Leadsom has quit the cabinet, saying she no longer believes the government’s approach will deliver Brexit.
Her resignation comes amid a backlash against Theresa May’s Brexit plan from Conservative MPs.
Several cabinet ministers have told the BBC that the PM cannot stay, with one saying it is “the end of the line”.
Mrs Leadsom previously ran for Tory leader but withdrew, allowing Theresa May to become prime minister.
In a letter to the prime minister, she said she did not believe “the UK would be a truly sovereign United Kingdom through the deal that is now proposed”.
She also described holding another referendum as “dangerously divisive”, and said she was opposed to the government “willingly facilitating such a concession”.
Mrs May has offered MPs a chance to hold a vote on another referendum if they back her Withdrawal Agreement Bill – the legislation needed to implement the agreement between the UK and EU on the terms of Brexit.