The firebrand senator edged out former deputy leader Kevin Hogan and Senate leader Bridget McKenzie in a partyroom ballot on Wednesday morning.
Darren Chester was elected the new deputy leader.
Mr Littleproud shocked colleagues as he announced he would step down from the regional party's top job on Tuesday with little notice, declaring he was "buggered" and didn't have the energy to continue as leader.
But the Queensland MP will continue representing his seat of Maranoa and has said he's willing to remain on the coalition front bench.
Mr Littleproud said he was feeling like a "million dollars" after stepping down, telling reporters on Wednesday morning he'd had his best sleep in eight months.
Consistently poor polling showing Pauline Hanson's One Nation overtaking the coalition in voter support has shaken the Nationals, who fear a wipe-out at the next federal election.
Seats in regional Queensland and NSW are considered particularly at risk, threatening a number of electorates held by the Nationals.
The rural party failed to win back the seat of Calare at the 2025 election to independent Andrew Gee, who quit the Nationals over their opposition to the Indigenous voice to parliament.
Firebrand Northern Territory senator Jacinta Nampijinpa Price also defected to the Liberal Party following the federal election defeat.
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