The 46-year-old Williams -- handed a wild card into the singles in New York some 29 years after reaching the final at Flushing Meadows as a teenager in 1997 -- exited 6-2, 7-6(6).
Williams, though, left with her head held high after a battling performance on the Arthur Ashe Stadium court that finished at just after 2am local time.
"It's not ideal," Williams said. "I would have preferred something else definitely. It wasn't great for either of us, so I think we both tried our best.
"I just don't know any other sport where you have to start that late. I definitely think there's room for improvement there.
"It's not easy to do in the first round. It's not easy to do it at 1:00 or 2:00 a.m. in the morning to try to find your rhythm and play your best, but I tried my best."
Kenin appeared to be cruising to a straightforward win after racing through the opening set and then taking a 4-2 lead in the second.
The 27-year-old American had a break point for what would have been a 5-2 lead but was then rocked by a ferocious fightback from Williams, who recovered to force a tie-break to the delight of the late-night crowd.
An improbable comeback appeared to be beckoning when Williams sprinted into a 6-2 lead in the tie-break, but Kenin regrouped impressively to rattle off six straight points to clinch victory.
Williams and Kenin's match got under way just after midnight local time following Novak Djokovic's marathon five-set loss earlier in the night session.
Williams, who saluted fans as she left the court, has given no indication of when she plans to call time on her singles career.
Her last victory in a Grand Slam singles game came at Wimbledon in 2021, where she reached the second round. Since then, Williams has made six Grand Slam singles appearances and exited in the first round on each occasion.
She will extend her US Open campaign when she plays in the women's doubles alongside sister Serena later in the tournament.
