He tweeted at Trump live on stage, saying he was "worried" about the president after the show had gone on for more than two hours.
First Kimmel wrote, "Hey @realDonaldTrump u up?" The crowd erupted in laughter.
Next, he made it a bit personal, writing, "@realDonaldTrump #Merylsayshi."
Kimmel was referring to Trump tweeting about the 20-time Oscar nominee after she spoke out about the president at the Golden Globe Awards.
Streep gave a speech at the Golden Globes, and though she never mentioned Trump by name, she said a "person asking to sit in the most respected seat in our country" gave an impression of a disabled reporter during one of his campaign stops.
He responded by tweeting, saying she was "over-rated."
"Meryl Streep, one of the most over-rated actresses in Hollywood, doesn't know me but attacked last night at the Golden Globes. She is a Hillary flunky who lost big. For the 100th time, I never 'mocked' a disabled reporter," he wrote.
Meryl Streep, one of the most over-rated actresses in Hollywood, doesn't know me but attacked last night at the Golden Globes. She is a.....— Donald J. Trump (@realDonaldTrump) January 9, 2017
Trump has yet to tweet back at Kimmel.