The 37-year-old was chasing a joint-record 24th grand slam title against Bianca Andreescu at Flushing Meadows but, as against Naomi Osaka last year and in consecutive Wimbledon finals, she came up short.
Williams turned in a nervy showing at Arthur Ashe Stadium, only briefly threatening to rally past her 19-year-old opponent late in the second set of a 6-3 7-5 reverse.
The match came 20 years on from Williams's first US Open victory and, asked about her incredible longevity after another painful defeat, she was hopeful there is still time in her career to return to winning ways in Major finals.
"I just feel really honoured to be out here. I'm just so proud I'm still out here competing at this level," she said at the post-match presentation. "It's not easy to be in this particular sport for 20 years.
"I have to give all thanks to Jehovah God for allowing me to have this moment to even be here.
"My team has been so supportive through all the ups and downs and downs and downs and downs and downs and downs...Hopefully we'll have some ups soon."
Williams has previously described herself as a fan of Andreescu and acknowledged that, with the exception of sister Venus, there is nobody she would rather lose to.
"Bianca played an unbelievable match. Congratulations," she said. "So proud and happy for you.
"It was incredible tennis out there. I wish I could have played better, but if anyone could win this tournament - outside of Venus - I'm happy it's Bianca."
Williams's only flicker of an opening in the match — a 23-minute stretch between Andreescu's first and second championship points — saw the crowd get involved, backing the American star.
She added: "I was just fighting at that point, trying to stay out there a little bit longer.
"Honestly, the fans started cheering so hard, it just made me play a little better and find a little bit more. I was really grateful for that."