The right-wing uprising that felled Mr. Kevin McCarthy marks the first time a US House of Representatives Speaker has ever lost a no-confidence vote.
The congressman was ousted as the head of the Republican majority in the lower chamber of Congress by a vote of 216 to 210.
After he reached an agreement with Senate Democrats to fund government agencies, hardliners in his party voted against him.
The management of the House Republican majority lacks an obvious successor.
To prevent a second potential government shutdown, Congress has just under 40 days to reach a resolution.
Republican Matt Gaetz of Florida, a supporter of Donald Trump, filed a motion to vacate on Monday night in order to remove Mr. McCarthy from his position.
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 politicians 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.”
Kevin McCarthy 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.
210 Republicans voted for him but eight voted against him in Tuesday’s vote, joining all Democrat members.
White House press secretary Karine Jean-Pierre said in a statement that President Joe Biden is hoping the House would quickly elect a new Speaker, and noted that the “challenges facing our nation will not wait”.
The last two Republican Speakers – Mr. Paul Ryan and Mr. 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.