Winfrey declared that King had seen injustice and "refused to look the other way."
"We, too, can be courageous by continuing to walk in the footsteps of the path that he forged," Winfrey said.
Two musicians who performed at the 1963 march also sang Wednesday. Peter Yarrow and Paul Stookey, from the trio Peter, Paul and Mary, sang Bob Dylan's "Blowin' in the Wind," backed by Sybrina Fulton and Tracy Martin, the parents of Trayvon Martin, whose 2012 shooting death sparked a national conversation about race. Mary Travers, the third artist in the group, died in 2009.
Obama's most personal remarks on race ahead of Wednesday's speech came in the aftermath of the July verdict that found Martin's killer not guilty.
In the crowd at the Lincoln Memorial, attendees used the occasion to remember where they were when they first heard King's "I have a Dream" speech.
"I grew up in a segregated environment. I never met a white person till I was a junior in college," said Betty Waller Gray, who traveled to Wednesday's march from Richmond. "It was just so emotional to be here today after knowing where I was in 1963. I was just a kid finishing high school back then."
Gilbert Lyons, an employee of the National Park Service, attended the original March on Washington half a decade ago and heard King utter his famous works in person.
"I went home with it in my head. I even spoke to my wife about it," he said. "It stayed with me. And the more I heard about Martin Luther King, the more things he was doing, I said, 'this man is great. He is a gentleman that can bring America back to themselves like they're supposed to be.' We're not supposed to be this race and that race. We are Americans."