The Juve loss came three weeks after their 49-match unbeaten record ended with a 3-1 home defeat by arch-rivals Inter Milan.
For AC Milan, the win completed an impressive week in which the Rossoneri also sealed qualification for the knockout stages of the Champions League.
An unconventional-looking front three of Robinho, Stephan El Shaarawy and Kevin Prince Boateng, pushed up from midfield, combined well to inspire's Milan's win.
Juventus found it difficult to deal with Milan's pressing play and went behind just before the half-hour mark when the ball appeared to strike Mauricio Isla's outstretched arm from Antonio Nocerino's header, although replays suggested it hit the Chilean's body.
Juve began to turn up the pressure later in the second half, with substitute Sebastian Giovinco, on for Fabio Quagliarella, going close with an overhead-kick from Simone Padoin's cross in the 62nd minute, but their lack of physical presence up front made life easier for the Milan back four.
It was a risky strategy and the home side lived dangerously at times, but worked hard to take all three points, putting a dent in Juve's title challenge and providing a possible platform for Milan to build on in the run up to the winter break.
Angelo Alessio, Conte's deputy on the touchline, bemoaned his side's failure to match Milan's work rate.
"When we don't play as a team, we suffer and we lose, like we did tonight," he told Sky Italia.
The hosts took the lead five minutes from the break after Alessio Cerci got himself on the end of a low Danilo D'Ambrosio cross, with Riccardo Meggiorini's dummy fooling the Fiorentina defence.
The visitors drew level from the penalty spot in the 75th minute after D'Ambros
