Almeria end Real's eight-match winning streak
A first-half goal by centre-back Juanito and a penalty from former Real Madrid reserve team striker Alvaro Negredo less than a minute after the break gave Almeria a memorable victory in their first season in the top flight in 27 years.
Real, whose previous league defeat was against Sevilla at the start of November, remain nine points clear of Barcelona at the top but the Catalans have a chance to cut the gap when they host Osasuna at the Nou Camp on Sunday.
Almeria, who have now won four in a row, moved up one place to seventh, three points ahead of Sevilla who visit Recreativo Huelva on Sunday.
"I'm not worried by this," Real coach Bernd Schuster told a news conference. "This setback comes at the perfect time for us and I'm sure we'll react well."
"This defeat just shows that this is the most competitive league and that's why it is the best."
Relegation-threatened Deportivo Coruna moved out of the drop zone after a 1-0 win at Real Betis in Saturday's late match, although the Galicians are only a point above Real Murcia and Recreativo Huelva who play on Sunday.
There was a frenetic start to the action at the Estadio Mediterraneo with Almeria and Real trading attacks in the opening quarter of an hour.
It was the home side, though, who appeared to have the clearer ideas as they broke rapidly down the flanks and looked to stretch the Madrid back four.
They took full advantage of a lapse by the Real defence to take the lead after 16 minutes, centre-back Juanito chipping into the net after picking up a rebound from a quick free kick.
Real also had several chances but Almeria's confidence remained undimmed and they got a second goal when Fabio Cannavaro conceded a penalty for pushing Negredo over in the area after a slick one-touch passing move.
Negredo, who joined Almeria from Real's reserve team Castilla at the start of the season, sent Iker Casillas the wrong way with his spot kick.
Raul almost pulled a goal back for Real with a close-range header but Alves produced a stunning one-handed save to turn the ball around the post.
The Brazilian made several more sharp stops to keep Real at bay while Almeria continued to create problems for their opponents with their speedy counter attacks.