Romelu Lukaku scored the winner from the penalty spot as Napoli ended Juventus' unbeaten start to the Serie A season.
Juventus, unbeaten in their first 21 league matches under new head coach Thiago Motta, took the lead through debutant Randal Kolo Muani.
League leaders Napoli equalised through Frank Anguissa, who headed in his 10th goal for the club in 140 appearances.
Napoli were awarded a penalty 20 minutes from time when Juventus defender Manuel Locatelli brought down former Manchester United midfielder Scott McTominay.
Lukaku, who saw a move to Juventus fall through in 2023, slotted his penalty past Michele di Gregorio for his ninth goal of the season.
The victory extends Napoli's lead over second-placed Inter Milan to six points, though they have played two games more than Simone Inzaghi's side.
Juventus' defeat leaves the club in fifth, 16 points behind Napoli and two points behind Lazio, who occupy the final Champions League spot.
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