Sesame Workshop, formerly known as the Children's Television Workshop, said after the campaign ad started running Tuesday it wanted it off the air.
"Sesame Workshop is a non-partisan, non-profit organization and we do not endorse candidates or participate in political campaigns," the organization said in a statement. "We have approved no campaign ads, and as is our general practice, have requested that the ad be taken down."
The spot, which was airing on national cable and broadcast TV during comedy shows, mocks a pledge Romney, the Republican presidential nominee, made in last week's debate to cut funding for the Public Broadcasting Service, even though he said he liked the 8-foot-2 primrose-yellow Big Bird character from "Sesame Street."
"I love Big Bird," Romney said. "But I'm not going to keep on spending money on things to borrow money from China to pay for."
The 30-second spot suggests Romney is targeting children's programming rather than legitimate threats to the economy.
"Big, yellow, a menace to our economy -- Mitt Romney knows it's not Wall Street you have to worry about, it's Sesame Street," an announcer says above ominous music.
It ends by showing Big Bird sitting in a basket and the announcer saying, "Mitt Romney -- taking on our enemies, no matter where they nest."
Romney spokeswoman Amanda Henneberg said: "Four years ago, President Obama said that if you don't have a record to run on, 'you make a big election about small things.' With 23 million people struggling for work, incomes falling, and gas prices soaring, Americans deserve more from their president."
PBS cuts would not kill Big Bird or any "Sesame Street" characters, Sesame Workshop Executive Vice President Sherrie Westin told CNN.
The show, which first aired Nov. 10, 1969, "receives very, very little funding from PBS," she said.