Cain didn't bring up the controversial facility in a campaign rally on Thursday, but told reporters afterward that he's concerned about the Islamic Center of Murfreesboro.
"It is an infringement and an abuse of our freedom of religion," he said. "And I don't agree with what's happening, because this isn't an innocent mosque."
The new mosque has been the subject of protests and counterprotests in the city about 35 miles southeast of Nashville.
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A county judge ruled in May that the mosque construction does not harm the residents who sued to try to stop it, but he allowed them to move forward on claims the county violated an open meetings law in approving it.
Opponents have used the hearings to argue that the mosque is part of a plot to expand Islamic extremism in the U.S. Cain appeared to agree.
"It is another example of why I believe in American laws and American courts," Cain said. "This is just another way to try to gradually sneak Shariah law into our laws, and I absolutely object to that."
As The Blaze reported back in March, Cain previously stirred controversy by saying that he would not want a Muslim bent on killing Americans in his administration.
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Cain spent much of the day making talk radio appearances, and his appearance in Murfreesboro drew hundreds to the courthouse square. Police diverted traffic to protect dozens who spilled out into the street.
Bill Ruark, of Tyler, Texas, said he decided to attend the rally after hearing Cain on the radio earlier in the day in Nashville, where Ruark's son is recording a country music video.
"He wants to define who are our enemies right now, and who are our allies," he said. "Morally, he's not afraid to say he's a Christian. He's not trying to force it down people's throats, and I appreciate that. And he's not afraid to say who he is."
Donah Hall, a postal worker in Murfreesboro, said she was impressed by Cain, but isn't sure about his electoral prospects.
"I'm just learning about him, and I was very pleased," she said. "I'm not sure come six months or nine months from now who I'll support.
"But it will definitely be a Republican, and not Barack Hussein Obama," Hall said. "I think he has just about broke our country up."