"The First Amendment protects freedom of religion. Senator Reid respects that, but thinks that the mosque should be built someplace else," the Nevada Democrat's office said in a statement.
Republicans have sharply criticized Obama since he expressed support Friday for the right to build the mosque and Islamic community center in lower Manhattan, citing the right to freedom of religion in America.
Reid faces a tough re-election race in November. ABC News reported he announced his opposition after Republican challenger Sharron Angle called on him to say whether he shared the president's view on the mosque, saying, "America is waiting."
The Hill said Reid, Obama's biggest ally in the Senate, became the highest-ranking Democrat to oppose the project, which would be built 2 1/2 blocks from the site where al-Qaida extremists crashed jetliners into the Twin Towers, killing nearly 3,000 people.
Reid urged Republicans to pass legislation to help emergency workers who responded to the Sept. 11, 2001, terrorist attack, Politico said.
"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," Reid spokesman Jim Manley said.
On Saturday, Obama said he had not been commenting on "the wisdom" of the Ground Zero project, only the right to build it.