Arda Turan struck in the second half to inflict Juventus' first defeat of the season and get Atletico Madrid's Champions League Group A campaign up and running on Wednesday evening.
The Turkish midfielder scored the only goal of the game in the 74th minute at the Vicente Calderon stadium to give last season's finalists all three points in the 1-0 victory.
After suffering a 3-2 defeat in their opener at Olympiakos a fortnight ago, Atletico went into the encounter without injured captain Gabi and under pressure to deliver.
Juve dominated possession but failed to test Atletico goalkeeper Miguel Moya throughout the match.
The outcome has lifted Atletico level on three points with Juve, Malmo and Olympiakos after two games played.
The visitors, heading into the game on the back of six straight wins in all competitions and having kept a clean sheet in the process, looked nervous in the early stages.
Raul Garcia headed Atletico's first chance just wide after five minutes before it was Juve who threatened, Claudio Marchisio seeing his long-range effort go off target.
Mario Mandzukic had the best chance to break the deadlock in the 25th minute but the Croatian's rising shot from 30 yards was kept out by Juve goalkeeper Gianluigi Buffon.
Shortly after, Diego Godin met a Koke free kick into the box with a header which went over the bar.
The hosts finished the first half with a good chance for Saul Niguez, whose header from Koke's cross flashed wide.
Simeone made his first substitution early in the second half with Antoine Griezmann replacing Niguez.
Juve struggled to break Atletico's defensive line and in the 72nd minute, Carlos Tevez' goal-bound attempt was blocked by a defender.
Atletico pressed forward more aggressively and were rewarded when Juanfran's cross found Turan in the box and his left-footed strike towards the far post beat Buffon.
The hosts almost shot themselves in the foot four minutes later when Stephan Lichsteiner's effort from 14 yards took a deflection off Garcia and the ball just went wide of the far post, with Moya already beaten, Atletico then holding on for a vital victory.