Barnaby Joyce has been sensationally returned to the Nationals leadership and deputy prime ministership, defeating Michael McCormack on Monday in a leadership spill.
The 21 members of the Nationals held a party room meeting on Monday morning in Federal Parliament where a motion to spill the leadership was put forward and carried.
The incoming Nationals leader is expected to address this media later on Monday.
Ag minister David Littleproud remained as deputy Nats leader.
Mr McCormack said briefly after the vote: "it's all democracy".
The vote will put pressure on Nats Mark Coultan and Darren Chester to hold their ministerial roles, as they are supporters of Mr McCormack.
Mr Joyce had downplayed that possibility ahead of the meeting, telling the Seven network, "there is no prospect of a spill at this time".
Mr McCormack went into the meeting standing firm in the face of the second challenge to his leadership in the past 18 months.
"If I survive, then the people who actually run against me, I think they should think long and hard about their futures, and I think they should think long and hard about the role they need to play in government and they should stop being so destabilising," he said.
Mr Joyce's last attempt to roll Mr McCormack in February 2020 ended in failure.
Mallee MP Anne Webster earlier told The Canberra Times the leadership speculation was a "disappointing distraction".
"Regional communities are not interested in the internal rumblings from Canberra," she said ahead of the meeting.
"They sent me here to advance their interests, that's what I'm doing. The Coalition has worked well together to bring Australia through the worst economic set back in 75 years. We need to continue to support our communities to recover from this pandemic."
Prior to the party room meeting, Nationals frontbencher Darren Chester, a staunch McCormack supporter, said country voters were sick of "bulldust" around leadership tensions.
He believed McCormack still had majority support.
"He should continue, in fact I think he'll lead us all the way to the next election," Mr Chester told reporters.
Queensland MP Michelle Landry was hopeful of a "normal" party room meeting.
"People just don't want to hear us talking about ourselves and having leadership challenges," she said outside Parliament House.
"I for one have had an absolute gutful of it all."
Outspoken Joyce backer Matt Canavan said he wouldn't ask for a return to the front bench under a change of leadership.
"There's a role for an independent voice in the Senate to play for regional Queensland in particular in my case," he told reporters.
While Liberal interventions in internal issues rankle Nationals, Prime Minister Scott Morrison threw his support behind Mr McCormack.
"I've got a wonderful partnership with Michael. We've worked very closely together and provided great stable leadership for Australia," he told 2GB radio.