Juventus' hopes of a fifth successive domestic double were extinguished after they were knocked out of the Coppa Italia by Atalanta in a 3-0 quarter-final defeat that marked their first domestic loss of the season and saw Massimiliano Allegri sent off.
The Bianconeri went into Wednesday's tie in Bergamo on the back of an extremely unconvincing performance in a 2-1 Serie A win over Lazio that sent them 11 points clear at the top.
And they were underwhelming again at the Stadio Atleti Azzurri d'Italia as they were undone by an Atalanta side that is the most prolific in Serie A, having scored 47 goals.
Gian Piero Gasperini's men dominated from the outset and Timothy Castagne's 37th-minute strike and Duvan Zapata's 19th goal of the season proved enough to set up a semi-final with Fiorentina, with the frustration that fuelled Allegri's dismissal after the second goal increased late on when a defensive mix up allowed Zapata to double his tally as their dominance of the competition ended.
Atalanta will have been buoyed by their stirring comeback against Roma on Sunday and Alejandro Gomez forced Wojciech Szczesny to turn behind with a fine effort from outside the box.
The hosts saw Josip Ilicic forced off through injury while Juve were dealt a significant blow when Giorgio Chiellini motioned to the bench that he could not continue.
It was his replacement, Joao Cancelo, one of the heroes of Juve's fightback at Lazio at the weekend, who was at fault for the opener. Cancelo was dispossessed by Castagne, who then surged towards the edge of the box and bent an effort beyond Szczesny and into the bottom-right corner.
And 125 seconds later Szczesny was beaten again in stunning fashion as the in-form Zapata turned and fired a fierce effort inside the Poland international's near-post, with a furious Allegri sent from the dugout in the aftermath after an exchange with the fourth official.
Juve improved after the interval and Daniele Rugani skimmed the woodwork with a header but they offered little thereafter and a terrible error by Mattia De Sciglio allowed Zapata to round Szczesny for his second as Allegri watched forlornly on from the stands.
What does it mean? - Juve invincible no more
While a first domestic loss for Juve is unlikely to provide hope for those vainly pursuing them in the title race, their inability to find the net and their sloppy play at the back will provide plenty of encouragement to an Atletico Madrid team that is extremely talented at both ends of the pitch ahead of their Champions League last 16, first leg next month.
Zapata reaches 20-goal mark
Though Zapata was aided by some questionable goalkeeping from Szczesny and some ponderous play from De Sciglio, the clinical manner in which he dispatched his double was indicative of a forward who has been one of the best in European football this season and now has 20 goals in all competitions.
Cancelo's Bergamo blunder
Pivotal to their revival in Rome, Cancelo's performance could not have been more contrasting to the one he produced at the Stadio Olimpico. He dallied on the ball for the opener and did more to hinder Juve's cause than help it on an evening he will want to quickly forget.
The visit of Parma is unlikely to provide a stumbling block for Juve in their seemingly inevitable march to the Scudetto. Seventh-placed Atalanta visit Cagliari as they push for the top four.