Kevin McCarthy has been toppled in a right-wing revolt – the first time ever that a US House of Representatives Speaker has lost a no-confidence vote.
The final tally was 216-210 to remove the California congressman as leader of the Republican majority in the lower chamber of Congress.
Ultra-conservatives mutinied after he struck a deal on Saturday with Senate Democrats to fund government agencies.
There is no obvious successor to oversee the House Republican majority.
Florida Republican Matt Gaetz, a Trump ally, filed a rarely used procedural tool known as a motion to vacate on Monday night to oust Mr McCarthy.
He accused the Speaker of making a secret deal with the White House to continue funding for Ukraine, amid negotiations to avert a partial government shutdown at the weekend. Mr McCarthy denies it.
At a private meeting of Republican lawmakers on Tuesday evening after losing his job, Mr McCarthy told colleagues he did not plan to run for Speaker again.
He later took aim at his political nemesis, Mr Gaetz, accusing him of attention-seeking.
“You know it was personal,” Mr McCarthy told a news conference, “it had nothing to do with spending.”
He said fundraising emails sent by Mr Gaetz amid the infighting were “not becoming of a member of Congress”.
The hardliners who ousted him “are not conservatives”, Mr McCarthy added.
He only became Speaker in January after a gruelling 15 rounds of voting in the chamber as Mr Gaetz and other right-wingers refused to support him.
Just eight Republicans voted to oust Mr McCarthy in Tuesday afternoon’s vote. He was able to muster support from 210 lawmakers – all Republicans.
But Democrats joined with the Republican defectors to remove the Speaker.
One vote against Mr McCarthy that surprised many came from a moderate Republican, Nancy Mace.
The South Carolina lawmaker said afterwards: “I am looking for a Speaker who will tell the truth to the American people, who will be honest and trustworthy with Congress, with both parties.”
Democratic House Leader Hakeem Jeffries had said in a letter to colleagues that he would not provide the votes needed to rescue Mr McCarthy.
Congresswoman Pramila Jayapal, a left-wing Democrat from the north-western US state of Washington, told reporters before the vote: “Let them wallow in their pigsty of incompetence.”
White House press secretary Karine Jean-Pierre said in a statement that President Joe Biden is hoping the House will quickly elect a new Speaker, noting that the “challenges facing our nation will not wait”.
The last two Republican Speakers – Paul Ryan and John Boehner – left Congress after repeated tangles with their more conservative colleagues.
The so-called motion to vacate had only previously been used twice in the past century to remove a Speaker – in 2015 and 2010 – though never successfully until Tuesday.