As Portman announced the nominees, she went off-script and said, "Here are the all-male nominees."
Her startled co-presenter, director Ron Howard, laughed nervously, and each of the nominated directors looked uncomfortable when the camera found them as their names were announced.
Sexism and inequality was the theme of the night, with stars wearing black and wearing pins supporting Time's Up, a legal defense fund for women who speak out against sexual harassment in the workplace, and numerous speeches addressing issues of inequality.
Only 7 percent of the 250 biggest domestic releases were directed by women in 2016, according to Variety.
Social media was loving Portman's call-out.
Later in the show, while presenting the award for Best Picture - Drama, Barbra Streisand, who won Best Director in 1984 for Yentl, said that it was unacceptable that she was the last and only woman to win that award.
"That was 34 years ago, folks," she said. "Time's up."