Judge Marco Aurelio Mello on Monday removed Mr Calheiros, who must face trial for alleged embezzlement.
Mr Calheiros says he will wait for a final decision by the whole Supreme Court on the issue on Wednesday.
Earlier, he had appealed against his removal, arguing that it could undermine a government austerity plan the Senate is due to vote on.
The controversial austerity plan, known as PEC-55, is scheduled to go to the Senate before the end of the year.
The judge's ruling was also a blow to centre-right President Michel Temer, a key ally of Mr Calheiros.
Under the order, Mr Calheiros would be temporarily replaced as Senate head by a member of the opposition Worker's Party, who may block the vote.
Judge Mello said his decision was valid with immediate effect, but could still be overturned by a majority of Supreme Court judges.
Senate lawyers said there was a legal basis for Mr Calheiros to continue until all the Supreme Court judges met to deliberate on Wednesday.
Mr Calheiros has been accused of taking bribes from a construction company.
Judge Mello approved the injunction requested by the left-wing Rede party which argued that a politician facing criminal charges cannot be in the presidential line of succession.
As head of the Senate, Mr Calheiros is second in line after the speaker of the Chamber of Deputies, Rodrigo Maia.
Brazil does not currently have a vice-president, as Mr Temer, who occupied the post, replaced Dilma Rousseff as president earlier this year.
She was dismissed after an impeachment trial.
The case against Mr Calheiros dates back to 2007.
He was forced to resign as Senate leader at the time, but was re-elected six years later. He denies wrongdoing.
Mr Calheiros is accused of agreeing with a construction company that it should pay maintenance fees for a daughter he had had during an extramarital affair with a journalist.
The company allegedly billed the Senate for the payments.
Mr Calheiros, 61, is also being investigated separately in connection with a big kickback scheme at the state-owned oil company, Petrobras.
Dozens of politicians, civil servants and businessmen have been detained since the investigation, known as Operation Car Wash, began in 2014.
Brazil's Senate leader Renan Calheiros has said he will defy a Supreme Court judge's order for him to stand down.