Williams, 37, is currently ranked 10th after making it to the finals at the US Open, but lost 6-2 7-5 to her 20-year-old compatriot.
The defeat means Williams’ quest to equal Margaret Court’s record of 24 Grand Slam singles titles continues.
“I’m so happy with this win – obviously you can tell with these emotions,” a crying Kenin told the crowd on Philippe Chatrier.
“Playing against Serena, you have to fight for every point.”
Williams’ defeat followed the shock exit by world number one Naomi Osaka a few hours earlier and leaves only three top-10 players in the women’s draw.
Earlier in the day, defending champion Simona Halep enjoyed a straightforward 55-minute victory, beating Ukraine’s Lesia Tsurenko 6-2 6-1 to reach the last 16.
World number 35 Kenin will play Australian eighth seed Ashleigh Barty in the fourth round, while Halep faces unseeded 18-year-old French Open debutant Iga Swiatek.
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