Andrea Leadsom, the leader of the House of Commons, has announced her resignation.
The move comes after Prime Minister Theresa May refused to resign despite mounting opposition to her latest Brexit deal.
Mrs Leadsom, a former Tory leadership candidate in 2016, said she had resigned "with great regret and a heavy heart".
Explaining her decision to step down on the eve of the European Parliament elections, she said: "I know there are important elections tomorrow, and many Conservatives have worked hard to support our excellent candidates.
"I considered carefully the timing of this decision, but I cannot fulfil my duty as Leader of the House tomorrow, to announce a Bill with new elements that I fundamentally oppose."
READ MORE: Theresa May 'clinging on' despite mounting opposition
Her letter of resignation went on to outline four reasons why she did not support the latest brexit deal.
She said: “I do not believe that we will be a truly sovereign United Kingdom through the deal that is now proposed.
"I have always maintained that a second referendum would be dangerously divisive, and I do not support the government willingly facilitating such a concession. It would also risk undermining our union which is something I passionately want to see strengthened.
"There has been such a breakdown of government processes that recent Brexit-related legislative proposals have not been properly scrutinised or approved by cabinet members.
“The tolerance to those in cabinet who have advocated policies contrary to the Government’s position has led to a complete breakdown of collective responsibility.”
Her resignation - the 36th of Mrs May's term in office - comes amid a backlash against the Prime minister's Brexit plan from Conservative MPs.
READ MORE: Cabinet ministers discuss PM's future
Several cabinet ministers said the Prime Minister could not stay, with one saying it is "the end of the line".
Labour said the resignation showed Mrs May's authority was "shot" and her time was up.
"While the Tories are ripping themselves apart, our country is in crisis," the party's chairman, Ian Lavery, said.
"The government has made a catastrophic mess of the Brexit negotiations, our steel industry is under threat and Universal Credit is pushing people into poverty.
"For the sake of the country, Theresa May needs to go, and we need an immediate General Election."
A Downing Street spokesman said: “Andrea Leadsom has served with distinction and great ability as a member of the government, and the prime minister is grateful for all of her work.
"We are disappointed that she has chosen to resign, and the prime minister remains focused on delivering the Brexitpeople voted for.”
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