Yesterday, he denied claims of sexual harassment against him as "spurious and defamatory" as he faces fresh questions about an 11-day road trip he undertook in December. "It has got to stop".
"It's quite evident that you can't go to the despatch box with issues like that surrounding you", he said.
Joyce said on Friday that none of the "litany of allegations" had been sustained.
Joyce was due to be the acting prime minister this week with Turnbull meeting US President Donald Trump in Washington, but opted to take leave.
Mr Joyce described the reporting as completely and utterly unwarranted, but found some humour in the situation.
The Nationals will need to vote on a new party leader, with Michael McCormack, Darren Chester and David Littleproud expected to be among the frontrunners.
The three men in line to replace Barnaby Joyce as Nationals leader include a gastroenterologist, a journalist and a rising star from the agriculture industry.
Reacting to the development, Prime Minister Malcolm Turnbull announced a ban on government ministers having sex with their employees and later called Joyce's actions a "shocking error of judgement".
The National Party will now elect a new leader, who will become deputy prime minister under the terms of the coalition agreement with Mr Turnbull's Liberal Party.
Now the 50-year-old is facing a sexual harassment allegation, with his party confirming a complaint had been lodged, reportedly by a West Australian woman.
"I thank Barnaby for his service as Deputy Prime Minister and in his various ministerial roles in which he has been a fierce advocate for rural and regional Australia", Mr Turnbull said.
The affair had brought an apparent rift between Turnbull and Joyce.
During the political sage over Joyce's personal life he was accused of hypocritical as he asked the public and the media to keep details of his personal life and relationships private. He is minister for veterans' affairs and minister for defence personnel.
"The party will greet this news with a heavy heart but we understand and respect his decision to stand down as leader", Mr Anthony said.
The resignation may relieve some pressure on Turnbull, whose ruling Liberal-National coalition has slumped further behind in opinion polls amid the scandal.
On Wednesday, Joyce told Fairfax Media he would ride out the storm but two days later he said it was "not hard at all" to decide to step down.