The Bianconeri were unbeaten in 22 league games and cruising heading into Sunday night's game at the Stadio San Paolo but goals from Jose Callejon and Dries Mertens gave Napoli the 2-0 victory.
Rafael Benitez's men may be a distant 17 points off the championship pace, yet Roma have a game in hand and could cut Juve's lead to only eight points by beating Parma on Wednesday.
The leaders will have 18-goal striker Carlos Tevez - suspended in Naples, leaving Juve rather toothless in attack - back for the run-in and ought to clinch enough wins from seven games to claim their third straight Scudetto.
Resounding jeers meant the hotly-anticipated match was finally under way and that Juve were on the ball, but Napoli would control the early stages.
A confident save from Gianluigi Buffon denied Callejon from close range in the sixth minute and, 60 seconds later, Marek Hamsik found the back of the net but was offside.
Buffon then made himself big to push Hamsik's first-time drive over the bar before Kwadwo Asamoah headed Faouzi Ghoulam's cross-cum-shot away from the far corner.
Juve finally rallied and tested home goalkeeper Jose Reina with a sighter from Stephan Lichtsteiner in the 26th minute.
Callejon bore down on Buffon once again and, eight minutes after watching the veteran stopper snaffle his attempted conversion on Lorenzo Insigne's cross, he broke Juve's creaking resolve.
Insigne again supplied the pass, which took a fateful deflection before Callejon ghosted in behind Asamoah to guide the ball beyond Buffon.
Dani Osvaldo's response to the setback was certainly ambitious - the Southampton loanee's acrobatic overhead kick flew into the stands.
Juve centre-back Giorgio Chiellini was lucky to avoid conceding a penalty just before the break when Gonzalo Higuain tumbled under his attentions on the by-line.
The visitors switched to a 4-3-3 formation shortly after the restart and soon began to enjoy more time on the ball in the final third.
Half-time substitute Mauricio Isla, on for Asamoah, beat the offside trap to cross low into the box, where Reina smothered, before Lichtsteiner sent the Spanish stopper sprawling to cover his effort.
But Napoli pushed back with confidence and soon got Buffon working again with a free-kick from Ghoulam that stung the Italy keeper's fingertips.
Andrea Pirlo's free-kicks have saved Juve in two recent matches but his latest attempt sailed harmlessly over the crossbar as an increasingly frustrated Antonio Conte prowled the touchline.
Osvaldo was unhappy, too, when his coach withdrew him for Mirko Vucinic, who almost made an instant impact when blasting wide of goal on the turn.
Napoli had waited patiently for Juve to run out of puff and broke forward again in the 78th minute.
Hamsik collected the ball from the left wing but, instead of beating Buffon with power, scuffed a weak shot against his legs.
It was the Slovakian's last contribution and his replacement Mertens would not make the same mistake.
The Belgian shielded the ball from Claudio Marchisio and promptly drilled a crisp strike into the top corner to confirm the visitors' fate.