Ciro Immobile struck twice and Paulo Dybala missed a penalty for the second Serie A game in succession as Lazio won 2-1 to hand Juventus a first home league defeat in 42 games.
Dybala's spot-kick woe came deep in stoppage time after referee Paolo Silvio Mazzoleni had used the Video Assistant Referee (VAR) system to rightly award Juve the chance to claim a point following a reckless challenge from Patric on Federico Bernardeschi.
But Lazio goalkeeper Thomas Strakosha dived low to his right to push Dybala's effort away before the final whistle went to condemn the Bianconeri to a first home defeat in Serie A since August 2015.
Juve's dominance at the Allianz Stadium has been one of the cornerstones of their six successive Serie A titles, but a growing sense that their Scudetto monopoly could be under serious threat will only increase after this eye-catching result.
The hosts had won their last six league meetings with Lazio to nil, and were on a revenge mission after their stunning stoppage-time defeat to Simone Inzaghi's side in the Supercoppa Italiana.
Douglas Costa opened his Juventus account, while Gonzalo Higuain bizarrely and inadvertently hit the bar from a yard out as Massimiliano Allegri's men dominated the opening half.
But Lazio had designs on upsetting the champions once again and former Juve youngster Immobile struck twice inside seven minutes after the break to turn the game on its head and put the visitors on course for their first league win over Juve in 14 years.
Both goals came as a result of creaking defensive play between Giorgio Chiellini and Andrea Barzagli and, having spurned a two-goal lead against Atalanta last time out - a game in which Dybala was foiled from 12 yards to snatch a win - questions may be asked of Allegri as Napoli's spectacular start to the season ramps up the pressure.
Allegri had opted to bench Dybala, despite the Bianconeri star failing to play a minute for Argentina during the international break, with Costa and Mario Mandzukic flanking Higuain in attack.
With five Lazio players strung in front of a back three, chances were at a premium for the hosts, but they converted their first clear-cut opportunity.
Sami Khedira's snapshot drew a fine save from Strakosha, only for Costa to snaffle the rebound - the goal upheld by VAR after replays showed Bastos had played him onside on the far side.
Strakosha denied Khedira more decisively with 10 minutes to play in the opening half, palming the German's blistering 30-yarder around the post.
The Allianz Stadium was collectively astonished when the score remained at 1-0 soon after when Higuain charged down Strakosha's clearance from no more than a yard out, only to see the ball incredibly rebound clear off the crossbar.
Lazio reached the interval having shown little sign of breaking their drought against the champions, but they pulled level just two minutes into the second half, Immobile curling beyond Buffon after he pulled away from Barzagli to latch onto Luis Alberto's pass.
Strakosha denied Higuain one-on-one as the striker's luckless game continued and Lazio stunned the home crowd soon after, Immobile again splitting the defence and rounding Gianluigi Buffon, who could only trip the striker.
Immobile picked himself up to send Buffon the wrong way and put the rarest of wins in the minds of Lazio fans.
Allegri called for Dybala as Juve's attempts to get back into the game came to nought and he was soon in the thick of the action, seeing a cross to fellow substitute Bernadeschi just hooked clear by Stefan Radu.
Dybala was denied by the post with a fizzing half-volley in the first minute of stoppage time, but looked set to be the hero when substitute Patric suffered a brain freeze with time almost up.
With the stage set, Dybala fluffed his lines again, Strakosha denying the South American to move Inzaghi's men level in the table with Juventus, who now look on anxiously as Napoli look to extend their advantage at the top against Roma.
