The superstar sisters, whose 14 Grand Slam doubles titles include two US Opens, rallied from a break down in the third set and led the deciding 10-point tiebreaker 5-0 but fell 6-3, 6-7 (6), 7-6 (7).
Venus and Serena hadn't played Grand Slam doubles since the 2022 US Open.
For Serena Williams, the 23-time Grand Slam singles champion considered by many to be tennis's greatest women's player, it was her first return to the courts of Flushing Meadows since then.
The 44-year-old had made a sensational return to major singles play as a Wimbledon wild card four years after "evolving away" from the game.
At the All-England club she lost to Joint in a Centre Court clash that captured the imagination of both diehard and casual tennis fans.
Even that experience didn't prepare Joint for the atmosphere on the cavernous Ashe Stadium.
The sisters received a thunderous ovation when they took court, all business in understated white outfits.
Every blistering volley and overhead smash from the Americans received raucous cheers, and when they sprinted to the lead in the decisive 10-point tiebreaker the noise was deafening.
"The atmosphere was surreal," Joint said. "Being able to play aginst such legends of the game is super special."
