The satirical spot, set to air on national broadcast and cable TV stations, shows images of convicted financiers, including Bernie Madoff and Enron's Ken Lay, and suggests Romney thinks Big Bird is behind their crimes.
"Only one man has the guts to speak his name," the ad says.
Romney said in last week's nationally televised debate with Obama that he liked Big Bird but would, if elected, end federal subsidies for the Public Broadcasting Service to help balance the budget. PBS airs "Sesame Street."
Obama seized on that comment after the debate and has used it to poke at Romney, and Tuesday's ad marked his re-election campaign's latest effort to make fun of his Republican rival.
But Sesame Workshop, which supports "Sesame Street," didn't see it as a laughing matter. It asked for the ad to be pulled.
"Sesame Workshop is a nonpartisan, nonprofit organization and we do not endorse candidates or participate in political campaigns," the organization said in a terse, two-sentence statement. "We have approved no campaign ads and, as is our general practice, have requested that the ad be taken down."
Obama campaign spokesman Ben LaBolt said the campaign was reviewing Sesame Workshop's concerns.
Romney's campaign countered that the ad shows Obama is focusing on inconsequential matters rather than urgent issues voters care about, like the economy and unemployment.
"Right now you've got 23 million Americans struggling to find work," spokesman Kevin Madden told reporters Tuesday. "I just find it troubling that the president's message, the president's focus 28 days from Election Day, is Big Bird."
PBS receives a portion of its funding from the Corporation for Public Broadcasting, which receives an annual appropriation from Congress. In 2012, CPB received $445 million in federal funding. PBS said in a news release after last week's presidential debate that public broadcasting receives about one one-hundredth of 1 percent of the federal budget.