The director, whose film BlacKkKlansman was nominated in the same category, waved his hands in disgust and seemingly tried to walk out of the Dolby Theatre.
According to Associated Press, Lee made his way to the building’s exit while the film’s producers were delivering their acceptance speeches, but was stopped by staff members.
Jordan Peele – who won an Oscar for Get Out last year – refused to clap the film.
Lee, whose film Do the Right Thing lost out to Driving Miss Daisy in 1990, said after the ceremony: “Every time someone is driving somewhere, I lose. They changed the seating arrangement.”
Green Book, which stars Viggo Mortensen and Mahershala Ali, has been widely criticised for falling foul to the “white saviour” trope. Its victory was a shock with many believing Netflix drama Roma would win the main prize.
The Peter Farrelly-directed comedy-drama also won Best Original Screenplay and Best Supporting Actor for Ali, who won the same award in 2017 for Moonlight.
Earlier in the evening, Lee won Best Adapted Screenplay for BlacKkKlansman.
Elsewhere, The Favourite star Olivia Colman won Best Actress shortly after Rami Malek took home Best Actor for his portrayal of Freddie Mercury in Queen biopic Bohemian Rhapsody. Mahershala Ali won Best Supporting Actor for Green Book, while Regina King won Best Supporting Actress for her performance in If Beale Street Could Talk.
The films vying for success at this year’s ceremony included Alfonso Cuarón's Netflix drama Roma, the Bradley Cooper-Lady Gaga musical A Star is Born and BlacKkKlansman. The former led with 10 alongside Yorgos Lanthimos’s The Favourite, with Adam McKay’s political satire Vice following close behind with eight.
You can find a full list of winners here.