Goals from Junior Firpo, Joaquin, Giovani Lo Celso and Sergio Canales were just about enough for Betis, for only their second win away from home this season.
Lionel Messi scored twice on his return, either side of a goal for substitute Vidal, but it wasn't enough as Barca slumped to their second loss in the league, and their first at home.
Barcelona have lost a game in which Lionel Messi has scored two or more goals for the first time ever in all competitions. Kryptonite.
The result means Barcelona don't extend their lead at the top. They're on 24 points from 12 games, with Atletico a point behind.
Meanwhile, the three points for Betis moves them onto 16 points, pushing them up to 12th in the table.