It's the latest in a long line of doubts raised by the Republican Party about the American leader's beliefs.
Trump's New York State campaign co-chairman Carl Paladino told a radio interview: "He is not a Christian."
"In the mind of the average American, there is no doubt he is a Muslim," he is quoted as saying by the Observer website.
"Look at what he's done with Iran, what he's done with the Sunni-Shia thing over in Iraq and Iran, and with ISIS."
Senior Republicans have been questioning Obama's faith since his election in 2008. They claim he was born to a Muslim man in Kenya.
President Obama is a practicing Christian who worshipped at Chicago's Trinity United Church of Christ before his election.
Donald Trump has, in the past, suggested that President Obama may not have been an American citizen.
It led to Obama releasing his full birth certificate.
