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Macklemore made his initial comments at Sheeran's New Jersey shows on Friday and Saturday as he introduced his 2024 protest song Hind's Hall, which condemns Israel's military action in Gaza.
After the rapper made his comments, the Israeli-American Council launched a petition urging Sheeran to remove Macklemore as his support act for the remainder of the tour.
The petition said Macklemore had "used the stage to deliver an extended political message, called for 'Free Palestine' and performed Hind's Hall while images from Gaza and other political messaging were projected across the stadium screens".
The council said the event was a concert, "not a political rally, not a protest, and not an activist event".
It added: "This becomes even more troubling when the message relies on selective imagery, biased messaging, and disputed claims presented to a massive audience as fact, without context or complexity."
Some ticket buyers posted messages addressed to Sheeran, external on social media criticising Macklemore's involvement in the tour, but the rapper also received praise and support from some fans, and his comments have received tens of thousands of shares.
On Monday, Macklemore uploaded a video, external on his social media feeds captioned: "Wanting all humans to be treated equal[ly] should never be controversial."
In the footage, he is seen saying: "One of the reasons I wanted to come on this tour was because I wanted to stand up on stages across America and say two words that are very near and dear to my heart: Free Palestine.
"I want those words to be loud and clear so the people from Gaza all the way to the occupied West Bank know that we have not forgotten them."
He concluded: "My message is for peace, love, for all human beings to be treated equally and with dignity."
BBC News has asked representatives for Ed Sheeran for a response to the calls for Macklemore to be removed as his support act.
Sheeran has been on his Loop tour in support of his latest album Play since January, but Macklemore only joined him as a support act recently as part of the US leg.
The rapper is also scheduled to join Sheeran at further US dates in the coming weeks.
In 2014, Macklemore apologised for a costume he wore on stage, which critics said was antisemitic.