Reid, who is locked in a tough reelection campaign, broke with President Obama in calling for the New York center to be built somewhere else. Obama on Friday endorsed the right of a group to build the center, which includes a prayer space, near Ground Zero.
"The First Amendment protects freedom of religion," Reid spokesman Jim Manley said in a statement. "Sen. Reid respects that but thinks that the mosque should be built someplace else."
Reid is the most senior Democrat to come out in opposition to the mosque.
Sen. Joe Lieberman (Conn.), an Independent who caucuses with Democrats, had questioned the wisdom of building the mosque, too. Other Democrats, like Rep. Keith Ellison (Minn.), the first Muslim member of Congress, have said that it's up to Democrats to support religious liberty.
The majority leader had been under pressure from his GOP opponent, Sharron Angle, to address the proposed mosque. Angle's campaign had said Reid "has a responsibility to stand up and say no to the mosque at Ground Zero or once again side with President Obama."
Angle's move reflects the desire by Republicans to make the controversy a national campaign issue.
Reid's spokesman used the mosque controversy to hammer Republicans who voted against a bill extending health benefits to first responders to the Sept. 11 attacks. That legislation stalled in Congress before lawmakers left for recess.
"If the Republicans are being sincere, they would help us pass this long-overdue bill to help the first responders whose health and livelihoods have been devastated because of their bravery on 9/11, rather than continuing to block this much-needed legislation," Manley said.