The clubs were separated by 40 points and 16 places coming into the match but it was third-bottom Valladolid who emerged victorious thanks to Fausto Rossi's first-half strike.
Second-placed Barcelona had several chances to get back into the match but could find no way past Valladolid keeper Diego Marino, and Gerardo Martino's men could now find themselves four points adrift of Real Madrid if the pacesetters beat Levante on Sunday.
"The team lacked ideas but not intensity. We completely failed in generating play and that's what stopped us from getting the result," said Martino.
"We lacked what makes us Barça: good elaboration in our build-up, finding spaces, breaking through on the wings and having depth. But we didn't lack attitude."
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