During his remarks, Killer Mike relayed a recent conversation with an activist named Jane Elliott, who, he said, told him: "A uterus doesn't qualify you to be president of the United States. You have to have policy that's reflective of social justice."
The comment ignited a debate on social media, with some accusing the rapper of being sexist –something he strongly denied through a series of posts on Twitter.
Sanders weighed in on Wednesday morning, with a spokesman saying he "doesn't believe gender should be a reason to vote for or against someone."
"That's the point Mike was making when he quoted Jane Elliott, the internationally known educator," said Sanders spokesman Michael Briggs. "We need to get beyond the gotcha politics and get to the issues at the heart of the election."
In a race featuring Hillary Clinton, who would be the country's first female president, gender has been an undercurrent, with several previous controversies emerging.
Killer Mike said on Twitter Tuesday night said that he was only repeating something he had been told and that he was supportive of other women politicians besides Clinton, including Nina Turner, a former Ohio state senator who also spoke on Sanders's behalf at the rally.
"I don't and never will Hate or think less of women," Killer Mike said in a tweet. "Sen. Nina Turner is my Next Great Political Champ but I'm sorry No HRC for me."
Elliott, whose words Killer Mike said he repeated, is a longtime anti-racism and gay rights activist.
In recent weeks, gender-related comments from Clinton supporters have also caused stirs. Ahead of the New Hampshire primary, former secretary of state Madeline Albright said at a Clinton event that "there’s a special place in hell for women who don’t help each other."
Killer Mike, an Atlanta-based rapper who is part of the duo Run the Jewels, has appeared with Sanders at several other events around the country and has sought to promote his candidacy, particularly to fellow African Americans.
In January, Killer Mike was among the Sanders supporters who represented the senator to reporters in the "spin room" at the Democratic debate in Charleston, S.C.
Sanders also visited a barber shop owned by Killer Mike, where a lengthy conversation between the two about racial justice and other issues was recorded and later released by the rapper in a series of widely viewed Web videos.