Juventus fell to a 1-0 loss at Sassuolo after a second-half goal by substitute Gregoire Defrel earned the points in Serie A on Sunday, condemning Massimiliano Allegri's side to successive league defeats for the first time this season.
Juve, seventh in the standings on 44 points, missed the chance to close the gap to the top six and remain four points behind Atalanta, who visit ninth-placed Fiorentina on Monday.
For Juventus, who took a small step towards the last four of the Europa League with a 1-0 home victory over Sporting in the first leg of their quarterfinal on Thursday, this was their first consecutive defeat in Serie A since October 2021.
Juve lost 2-1 at Lazio in their last league outing.
After a cautious start on Sunday, the match sprung to life when Sassuolo midfielder Maxime Lopez tested Juventus goalkeeper Mattia Perin with a header in the 55th minute.
A minute later Perin pulled off another brilliant save to keep out Defrel's towering header following a corner.
However, the halftime substituteeventually broke the deadlock in the 64th minute, firing inside the right post on the rebound following a corner.
Juve came close to finding an equaliser nine minutes later through Adrien Rabiot who leapt inside the box to connect with Angel Di Maria's corner but keeper Andrea Consigli superbly turned the ball over the bar.
Di Maria, who replaced Arkadiusz Milik just before the hour, had a chance 15 minutes from time when he met a sublime cross from Federico Chiesa but narrowly missed the target.
Allegri brought on Paul Pogba in the final 10 minutes for the Frenchman's third league appearance of an injury-plagued season but the Turin side did not come any closer to scoring.
Juventus, for whom winning the Europa League remains their most likely route into next season's Champions League, face Sporting in Lisbon in their Europa league return leg on Thursday before hosting runaway Serie A leaders Napoli next Sunday.
Sassuolo, who moved up three places to 10th on 40 points from 30 games, travel to Salernitana on Saturday.
Sixteenth-placed Lecce, five points above the relegation zone, drew 1-1 at home to Sampdoria who are rooted to the bottom of the table 10 points adrift of the safety line, while Torino, in 11th, had the same scoreline against 15th-placed Salernitana.