Gingrich's campaign on Wednesday said Alabama and Mississippi are must-wins for the House former Speaker.
The Red, White and Blue Fund argues Santorum would have a shot at beating Romney if Gingrich would withdraw from the race.
"With Gingrich exiting the race it would be a true head-to-head race and conservatives would be able to make a choice between a consistent conservative in Rick Santorum or Mitt Romney," Roy said in a statement on Wednesday. "For instance, with Gingrich out of the race, Santorum would have won both Ohio and Michigan. Newt has become a hindrance to a conservative alternative."
Gingrich has rejected calls for him to drop out.
"We are staying in this race because I believe it is going to be impossible for a moderate to win the general election," Gingrich said at a campaign event in Alabama, according to the Atlanta Journal-Constitution.
Watch the super-PAC’s healthcare ad here.
In response to the ad, Romney's campaign accused the Santorum campaign of twisting and exaggerating the former Massachusetts governor's record..
"Sen. Santorum and his allies have a habit of making distortions, exaggerations and falsehoods about Mitt Romney’s record while at the same time denying Sen. Santorum’s well-documented lobbying past," Romney spokeswoman Andrea Saul said in a statement. "Voters are tired of the same political double-speak and understand it’s going to take an outsider to change the status quo in Washington."
—This story was updated at 12:52 p.m.