Makoni will run against Mugabe as an independent in the presidential election.
"He has expelled himself from the party, he was not expelled by anyone but himself. That is the position according to the rules of the party. So yes, indeed, he is expelled from the party," Emmerson Mnangagwa, Zanu-PF's secretary for legal affairs, told Reuters.
Makoni had been expected to register for the poll on Friday but Zimbabwean authorities postponed the nomination date to February 15 after some politicians won a court order to delay the process.
On Wednesday, Zimbabwe's government-controlled media branded Makoni a British and American-sponsored puppet seeking to split the Zanu-PF and oust Mugabe.
Joseph Chinotimba, deputy leader of the war veterans, was quoted as saying that Makoni was a political turncoat who would suffer a humiliating defeat in the March 29 general election.
"We are now going to campaign vigorously for President Mugabe. I feel sorry for Makoni, he has lost the political plot," Chinotimba told the Herald newspaper.
"From today to the nomination date we will have finished with them. Traitors should know that Zanu-PF has a history of dealing harshly with their kind."
"We are calling on all war veterans to take control of the party," he said.
War veterans, who fought in the 1970s conflict, along with members of Zanu-PF, have turned violent in the past in support of Mugabe. There are 30,000 war veterans in Zimbabwe.
Zimbabwe's privately owned media have reported that Makoni is backed by a faction led by Solomon Mujuru, a retired army general and member of the ruling Zanu-PF party's top decision-making body.
Despite economic turmoil, 83-year-old Mugabe is expected to defeat the divided opposition, although it is not yet clear how much support Makoni will receive.
Analysts say shortages of food, foreign currency and fuel, and the world's highest inflation rate, officially pegged at 26,000 per cent, are the biggest challenge to Mugabe's rule.
But the opposition has failed to capitalise on Mugabe's failure to ease the crisis and it remains to be seen if daily hardships will push Zimbabweans to support Makoni.