The MP for Orpington revealed in an open letter he would vote against the withdrawal agreement in the Commons.
The Tory MP, who headed Parliament's fight to give MPs the final say on Theresa May's Brexit deal at the end of the negotiations, claimed Parliament could impose a proposal to extend Article 50 at the end of the negotiations in case MPs voted down the deal reached by the Prime Minister with Brussels.
For some time, Jo Johnson has struggled with the unfolding reality of Brexit.
"To present the nation with a choice between two deeply unattractive outcomes, vassalage and chaos, is a failure of British statecraft on a scale unseen since the Suez crisis", he said.
Britain's junior Brexit minister told a German media outlet he was optimistic that Britain and the European Union would reach a Brexit deal soon, adding that he hoped this would happen in the next three weeks.
Boris Johnson, who himself resigned over May's Brexit plans, backed his brother's decision, saying: "We may not have agreed about Brexit but we are united in dismay at the intellectually and politically indefensible of the UK position".
His brother praised his decision, saying they were "united in dismay" at the PM's handling of the negotiations.
"The referendum in 2016 was the biggest democratic exercise in this country's history".
"This is the time for all of them, as Jo Johnson has done, to look deep inside their consciences, to reflect deeply on their responsibilities, and do the right thing for future generations".
'On this most crucial of questions, I believe it is entirely right to go back to the people and ask them to confirm their decision to leave the European Union and, if they choose to do that, to give them the final say on whether we leave with the Prime Minister's deal or without it'.
"We will not under any circumstances have a 2nd referendum".
The MP, who is Boris Johnson's brother, said the withdrawal deal now being negotiated with the European Union "will be a awful mistake".
Mr Johnson's sister Rachel said she was "hugely proud" of her "honourable and principled brother Jo who has put the interests of the country ahead of his political career".
Cabinet ministers have been visiting 10 Downing St this week to see details of the government's plan for the "divorce deal" and subsequent trading and customs relationship with the EU.
Barnier is now attempting to negotiate a deal with Prime Minister Theresa May's government for Britain's "orderly" exit, which will take place in March next year.
As campaigners step up pressure on the government calling for the public to be given another say on Brexit, the opposition Labour Party leader Jeremy Corbyn dashed campaigners' hopes he could back another referendum.
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