The 51-year-old New York City native had previously won for The Pianist, when he became the youngest best actor winner at age 29.
Brody joins an elite group of multiple winners in this category that includes Daniel Day-Lewis, Tom Hanks, Anthony Hopkins, Jack Nicholson and Spencer Tracy.
Brody has said his mother escaped from Hungary and moved across the Atlantic, echoing the journey of the character he plays, a modernist architect named Laszlo Toth.
"I understand a great deal about the repercussions of that on her life and her work as an artist," he told reporters at the Venice Film Festival. Brody's mother is the celebrated photographer Sylvia Plachy.
Sean Baker won the Academy Award for best director for Anora, a film about an exotic dancer and sex worker who has the chance of a Cinderella story when she marries the son of a Russian oligarch.
Baker had been favoured to win the directing award, having collected top honours from the Directors Guild of America and the Producers Guild of America, and the directing award at the Film Independent Spirit Awards.
Earlier Zoe Saldana was named best supporting actress for her role as the fixer for a Mexican drug lord in the Spanish language Netflix musical Emilia Perez while Kieran Culkin won best supporting actor for playing one of two cousins who travel to Poland to study their family's history in A Real Pain.
He beat Australia's Guy Pearce, who was nominated for his performance in The Brutalist.
"I have no idea how I got here," Culkin said on stage at the Dolby Theatre in Hollywood.
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