Four days on from its stunning comeback win over Manchester City in the UEFA Champions League semi-finals, Madrid made seven changes for its trip to Wanda Metropolitano.
That told as Madrid, which was crowned Spanish champion last week with four games to spare, fell behind to a 40th-minute Yannick Carrasco penalty that was awarded after a VAR check.
Despite the introduction of Luka Modric and Vinicius Junior, Madrid could not find a leveller in the absence of top scorer Karim Benzema as it fell to a rare El Derbi defeat.
Much was made of Atleti's decision to not give Madrid a guard of honour ahead of kick-off in their neighbours' first league outing since sealing a 35th league crown.
The home side were far more fired up for the match and twice went close in the opening eight minutes, with Angel Correa and Carrasco firing wide from good positions.
Carrasco made the breakthrough from the penalty spot before half-time, though, after Matheus Cunha was barged over by Jesus Vallejo just inside the box.
The referee had initially allowed play to continue for a couple of minutes, but he was instructed by the VAR to check the pitchside monitor and reversed his initial decision.
Luka Jovic was denied by Jan Oblak and Casemiro should have done far more with a tame shot that was curled straight at the Atletico keeper as Madrid pushed for a leveller.
Atleti wasted chances of its own as Carrasco fired over and Cunha could not convert past Andriy Lunin when played in by substitute Antoine Griezmann, who also went close.
Carrasco nearly sealed things when hitting the post 14 minutes from time, but his earlier penalty proved enough in the end thanks to another Oblak save to keep out Vinicius and a bad miss from Nacho.