Juventus, who is yet to recover from its loss after Sunday’s tough league game to which they lost their first Serie A match of the season, 0-2 at Genoa.
Stefano Sturaro, who joined Genoa on loan from Juventus in January before the deal was made permanent, put the home side in front in the second half.
Then, fellow substitute Goran Pandev secured the shock result with 10 minutes remaining.
Cristiano Ronaldo inspired a remarkable European comeback for the Italian champions on Tuesday when he scored a hat-trick to overturn a 2-0 first leg defeat against Atletico Madrid.
The victory had sent his side through to the quarter-finals with a 3-2 aggregate win.
However, the Portuguese was left out of the matchday squad for the trip to the Stadio Luigi Ferraris.
Meanwhile, Juventus coach Massimiliano Allegri was left frustrated as Genoa took points off his side for the second time this season, having drawn 1-1 in Turin in October.
“I expected a different Juventus,” Allegri told Italian broadcasters.
“We did not put in a good performance. We made some mistakes and then this goal came out of nowhere.
“Much like the Coppa Italia defeat to Atalanta, we picked the right game to lose, because it’s just before the break for international duty. So, we have time to unplug and rest.
“I am actually happy for Stefano, because if anyone had to score against us, I’m glad it was Sturaro.
“He’s a lovely guy and suffered so much, out of action for practically a year with that tendon injury. So, he deserves it.”
Juventus had dropped points just three times this season going into the game, with draws against Genoa, Atalanta and Parma.
They were unbeaten overall in the top-flight for the last 31 rounds, since losing 0-1 to Napoli on April 22, 2018.
However, the hosts started the brighter as Christian Kouame forced a good save from former Genoa goalkeeper Mattia Perin, before Cesare Prandelli’s side were awarded a penalty kick for handball.
But a Video Assistant Referee (VAR) review overturned the decision.
Juventus thought they had gone in front after the break when Paulo Dybala swept home a finish, but VAR chalked the goal off for an offside in the build-up.
Sturaro then curled a finish into the corner against his parent club just minutes after coming on, before Pandev made sure of the three points with a powerful finish.
In spite of the result, Juventus remain 18 points clear of second-placed Napoli at the summit of the table, while Genoa climbed above Sassuolo into 12th place on 33 points.