The two-time Oscar winner made the comments while hosting a fundraiser for US President Barack Obama's re-election campaign at the Greenwich Hotel in New York City on Monday (March 19).
De Niro took the opportunity to joke about the wives of US Republican candidates Mitt Romney, Rick Santorum and Newt Gingrich prior to introducing the first lady.
"Callista Gingrich. Karen Santorum. Ann Romney. Now do you really think our country is ready for a white first lady?" the legendary actor quipped.
Michelle Obama's press secretary has since condemned the joke to CNN, calling the remark "inappropriate".
The remark was also branded "inexcusable" by candidate Newt Gingrich and "sad" by Rick Santorum.
De Niro apologised for the joke in a press statement issued on Tuesday evening.
"My remarks, although spoken with satirical jest, were not meant to offend or embarrass anyone, especially the first lady," the actor said.
The US First Lady was in New York City this week for her first-ever appearance on The Late Show with David Letterman.
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