Djokovic, whose last first round defeat at a grand slam came in the 2006 Australian Open, went up two sets to one and a break in the fourth set against Navone. However, he was unable to close it out against the world number 49, who won 11 of the last 12 service games to complete the upset.
The first set went Navone's way as he held his nerve in a tiebreak. Both players broke the other once each, with Djokovic even holding a mini-break during the tiebreak. Yet, the Argentinian won the final two points to claim the opening set in 75 minutes.
The 39-year-old, seeded fourth for the tournament, responded by winning the next two sets. He broke Navone's serve in the penultimate game of both sets to go up two sets to one.
Djokovic struggled in the third set, throwing up on a couple of occasions as he struggled on court. Taking a 2-1 lead in the fourth set, he could not keep up the level needed to get over the line.
Navone proceeded to break the Serb right back before winning the next four games to force a fifth set. Losing the first three games of the deciding set, Djokovic wiped tears from his eyes as he won what would be the last game before his opponent finished off the match.
With the result, Navone moves on to face the winner of Matteo Berettini and Stan Wawrinka in the next round.
For Djokovic, who had 70 unforced errors in the match, the defeat marked his earliest-ever exit in Flushing Meadows. He will finish the year without a grand slam title for the third consecutive year. Since his first major win in 2008, he had previously never went more than two straight years without a slam victory.