Trump and other pageant leaders said Prejean was being sacked not because of the remarks but because she hadn't been holding up her end of the agreement she signed when she entered the pageant.
"Carrie is a beautiful young woman and I wish her well as she pursues her other interests," Trump said.
Prejean spokeswoman Melany Ethridge declined to comment, saying she hadn't been able to reach the former beauty queen since the announcement of her ouster.
"This was a decision based solely on contract violations, including Ms. Prejean's unwillingness to make appearances on behalf of the Miss California USA organization," the California pageant's executive director, Keith Lewis, said in a statement.
He had complained at a news conference last month that Prejean was skipping Miss California USA events while speaking out against gay marriage at unsanctioned appearances.
After the complaints reached Trump, who owns Miss California USA's parent organization, the mogul decided to give her a second chance and keep her on. On Wednesday, he changed his mind.
"I told Carrie she needed to get back to work and honor her contract with the Miss California USA Organization and I gave her the opportunity to do so," Trump said in a statement. "Unfortunately it just doesn't look like it is going to happen."
Pageant officials said Prejean would be replaced by the Miss California pageant's first runner-up, Tami Farrell, 24, who was crowned Miss Teen USA in 2003. They had announced last month that Farrell was being named a special ambassador to fill in for Prejean at any events she didn't attend.
During the Miss USA competition, Prejean was first runner-up after answering a judge's question about gay marriage by saying wedding vows should be reserved for men and women.
"No offense to anybody out there, but that's how I was raised," she said.
After the response drew heavy criticism she began speaking to crowds opposed to gay marriage, violating a section of her contract that required she clear unsanctioned appearances with pageant officials. About the same time, seminude photos of her began to surface on the Internet.
Although the photos were mild, pageant officials said she also violated her contract by not disclosing their existence to them.
When Trump initially decided to keep Prejean on, Lewis' co-executive director, former Miss USA Shanna Moakler resigned in protest.
"My faith has been restored in the Miss Universe Organization and with Donald Trump," Moakler said in a statement after Wednesday's announcement. She didn't say whether she planned to return to the organization.
"I think she's a young girl, and I think she was thrown into the spotlight (after the Miss USA pageant)," Moakler told The Early Show last month. "I think she was not prepared. I think a lot of organizations latched onto her. They exploited her. They didn't prepare her. I think, you know, she's very honest when she says she never expected this."