The former London mayor’s decision not to join the battle leaves Home Secretary Theresa May as hot favourite to be the next Prime Minister.
It came after the shock announcement by fellow Brexit campaigner Michael Gove - who had widely been expected to be Mr Johnson’s running mate - that he was putting himself forward for the leadership.
In a dramatic press conference just moments before the deadline for nominations passed, Mr Johnson said that the next Tory leader would have to unify his party and ensure that Britain stood tall in the world.
And he said: “Having consulted colleagues and in view of the circumstances in Parliament, I have concluded that person cannot be me.”
Earlier, Mr Gove said: “I have repeatedly said that I do not want to be prime minister. That has always been my view. But events since last Thursday have weighed heavily with me,” he said.
“I respect and admire all the candidates running for the leadership. In particular, I wanted to help build a team behind Boris Johnson so that a politician who argued for leaving the European Union could lead us to a better future.
“But I have come, reluctantly, to the conclusion that Boris cannot provide the leadership or build the team for the task ahead.
“I have therefore decided to put my name forward for the leadership. I want there to be an open and positive debate about the path the country will now take. Whatever the verdict of that debate I will respect it. In the next few days I will lay out my plan for the United Kingdom which I hope can provide unity and change.”
The declaration is a devastating blow for Mr Johnson, who was expected to stand with the backing of the Justice Secretary.
Mr Johnson will use his launch speech to present what was described as a “positive, optimistic vision” of Britain outside the EU, offering “a chance to believe in ourselves”.
Stephen Crabb, Liam Fox and Andrea Leadsom have also been confirmed as leadership contenders.
Theresa May formally launched her campaign this morning with a promise that she can offer “leadership that can unite our party and our country”.
The Home Secretary is hot favourite to make it onto the ballot paper alongside Mr Johnson in the final run-off vote.
Mrs May - who will fight on her record as a hard-headed and serious long-serving holder of one of the Cabinet’s most difficult posts - hit out at her probable main rival with a newspaper article in which she warned that politics is “not a game”.
Setting out her proposed programme in a speech in London, Mrs May - who was a low-key supporter of Remain during the referendum - made clear she will not attempt to back away from last week’s vote to leave the EU, saying “Brexit means Brexit”.
But she said she would not kick-off the two-year process of negotiating withdrawal until the UK’s negotiating strategy is agreed - probably not before the end of this year. She said that she would create a new Government department, headed by a Cabinet-level minister who had campaigned for Leave, to oversee the UK’s departure from the EU.
She said she would not order an emergency budget in response to the Brexit vote and would not call a snap election ahead of the scheduled date of 2020.
Mrs May said: “Following last week’s referendum, our country needs strong leadership to steer us through this period of economic and political uncertainty and to negotiate the best possible terms as we leave the EU.
“We need leadership that can unite our party and our country.”
Page 2: 09:55
With Labour “tearing itself apart” and Scottish Nationalists mooting a second independence referendum, Mrs May said it was “nothing less than the patriotic duty of our party to unite and to govern in the best interests of the whole country.
“We need a bold new positive vision for the future of our country - a country that works not for a privileged few but for every one of us.”
In a clear swipe at Mr Johnson, Mrs May said politics was not a “game”.
She said: “If you are from an ordinary working class family, life is just much harder than many people in politics realise.
“You have a job but you don’t always have job security, you have your own home but you worry about mortgage rates going up, you can just about manage but you worry about the cost of living and the quality of the local school because there is no other choice for you.
“Frankly, not everybody in Westminster understands what it’s like to live like this and some need to be told that it isn’t a game. It’s a serious business that has real consequences for people’s lives.”
A YouGov poll for The Times of 1,000 Conservative Party members put Mrs May ahead on 36% with Mr Johnson on 27%. If it came down to a straight two-horse race between the pair, the advantage for Mrs May was even bigger at 55% to 38%.
The entry into the race of the two candidates seen as the likely front-runners follows announcements by former defence secretary Liam Fox and the self-styled “underdog”, Work and Pensions Secretary Stephen Crabb, that they were putting themselves forward.
Health Secretary Jeremy Hunt and Education Secretary Nicky Morgan, who have also both indicated they could stand, have until the close of nominations at midday to make up their minds.
Dr Fox, who campaigned for Leave in the referendum and who ran against Mr Cameron in the 2005 leadership election, will hope to draw support from the right of the party in the battle to succeed him.
The former cabinet minister resigned in 2011 after being found guilty of breaching the ministerial code over his links with self-styled adviser Adam Werritty, whom he met 40 times in the Ministry of Defence and on trips abroad.
A series of run-offs will begin on Tuesday as MPs whittle down the hopefuls to two, who will tussle for the support of grassroots members in a one-member-one-vote contest.
Speaking to Good Morning Britain, Dr Fox said any candidate who had voted to stay in the EU would struggle with credibility.
He said: “I think it’s much harder for anybody who voted Remain to have credibility amongst the voters who voted to leave in terms of leading the country. It’s not impossible, but it’s much harder.
“For those of us on that Leave side there is a natural credibility with those voters.”
Asked who he would support if he dropped out of the race, he said they would need to be “clear about leaving the European Union and not betraying what the British people told us”.
He added: “I’m a very good friend of Stephen Crabb, I had great fun campaigning with Boris Johnson on the winning side, I have great experience working with Theresa May in government on national security. I respect all of them and I hope that’s very much the tone.”