The film includes footage of Trump after the violent white supremacist protests in Charlottesville, Virginia.
Lee did not directly name Trump.
He spoke about black history and his family history, saying his grandmother's mother was a slave, before stressing the presidential election next year.
Said Lee: "Let's all be on the right side of history. Make the moral choice between love versus hate."
Lee's Oscar win was his first ever — a long-awaited achievement in a film career spanning decades. He was also nominated for Best Director.
An ecstatic Lee jumped into presenter Samuel L. Jackson's arms when he took the stage.
According to the Post, previously, the Brooklyn-raised Lee had only received a Best Screenplay nod for Do The Right Thing in 1990 and a Best Documentary Feature nomination in 1998 for 4 Little Girls.
He won neither, but received an honorary award in 2015 for being "a champion of independent film and an inspiration to young filmmakers."
When Ryan Seacrest asked Lee, during E!'s red carpet coverage before the ceremony, if those slights were "water under the bridge," the director replied, "I wouldn't go that far."
BlacKkKlansman was also nominated for Best Picture, Best Supporting Actor (Adam Driver), Best Director (Lee), Score and Editing.