The moment came during a segment that analyzed the New York politician's accent.
The panel had just played a clip of Ocasio-Cortez speaking to attendees at a rally in Memphis with what some said was a manufactured Southern drawl.
'We cannot afford to leave Tennessee behind,' she told attendees gathered to support US House candidate Justin Pearson, before saying the same for states like Georgia and South Carolina.
Gutfeld took the segment a step further while simultaneously lampooning a line from the 36-year-old's speech.
'She mentions behind six times. And all I’m thinking about is her behind!' Gutfeld, 61, riffed, after reading the quote in full.
A noticeable lull followed, as Gutfeld waited for the off-color joke to land.
Greg Gutfeld made a joke about Congresswoman Alexandria Ocasio-Cortez's 'behind' on Monday after her appearance at a rally for a fellow progressive seeking to join the House of RepresentativesThe quip received little reaction from on the comic's co-panelists, three of whom were women
The panel - rounded out by Harold Ford Jr, Dana Perino, Kennedy, and Fox & Friends host Charles Hurt - remained mostly silent.
A light chuckle from one of the two male panelists was heard off-screen.
The Daily Mail has approached Representative Ocasio-Cortez and Fox News for comment.
The congresswoman has yet to respond to Gutfeld's remarks.
'He has sexualized me on his show. He has sexually harassed me on his show. He has engaged in horrific, sexually exploitative rhetoric,' Ocasio-Cortez said of The Five's Jesse Watters, during an encounter with a Fox News producer in DC in January.
She had been asked if she would 'join' the conservative pundit on either of his programs - The Five and Jesse Watters Primetime - at some point in the future.
AOC previously refused to join Fox News for an interview after accusing Jesse Watters of sexually harassing her in January
'So why don't you tell me what you think is acceptable to tell a woman?' she told the producer, labeling Watters a 'pervert.'
Gutfeld and Watters are two of The Five's most recognizable co-hosts.
Since launching in 2011, according to Nielsen, the program has grown its audience by 161 percent, from 1.5 million to more than 3.7 million.
It is one of the most-watched daily cable news programs among Democrats, reaching 765,000 viewers registered to the party, according to Nielsen - more than CNN and MS NOW combined.
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