The ad jokingly accuses Romney of killing corporations while an executive at private-equity firm Bain Capital, and reasons that since Romney has previously likened corporations to people, he is therefore a "serial killer."
"Mitt Romney says he's for corporations, but Mitt Romney has a secret," says the narrator in the ad. "As head of Bain Capital he bought companies, carved them up and got rid of what he couldn't use. If Mitt Romney really believes 'corporations are people, my friend' than Mitt Romney is a serial killer. He's Mitt the Ripper."
When asked about the "killer" ad by George Stephanopoulos on ABC's "This Week" Sunday, Colbert denied having previously seen the spot.
"I had nothing to do with that ad. I have no control over that ad," Colbert said. "I don't know if Mitt Romney is a serial killer. That's a question he's going to have to answer."
Candidates are forbidden from coordinating with super-PACs, but many are run and funded by former campaign staff, close friends or family of the candidate, an issue Colbert has raised repeatedly on his Comedy Central show, "The Colbert Report."
Colbert announced that he would give up control of his super-PAC to fellow comedian Jon Stewart in order to explore a presidential run on Thursday night's episode of his show.
Observers see Colbert's mock run as an attempt to highlight issues about money in politics. The comedian has stayed in-character during interviews and insisted that his run is not just for laughs.
However, it is too late to appear on the ballot in the Palmetto State and there are no write-in candidates, according to Matt Moore, executive director of the South Carolina Republican Party.
"Just because something's difficult doesn't mean it shouldn't be worth doing. I'm exploring right now. I'm a one-man Lewis and Clark and I'm just looking for my Sacagawea," said Colbert on ABC News.
The South Carolina primary takes place Saturday, Jan. 21.
For more on Colbert's super-PAC and presidential exploratory committee, click here.
—Brendan Sasso contributed