The champions had defeated Almeria 4-1 last week but the score flattered Gerardo Martino's men after a sluggish display and they were even worse at Estadio Jose Zorrilla on Saturday.
Valladolid goalkeeper Diego Marino produced a good performance to continually deny Lionel Messi, while Neymar missed a great chance in the second half.
Fausto Rossi's 17th-minute strike was enough for the hosts, as the Italian pounced on a loose ball in the area following a kind ricochet.
Barca attempted to pile on late pressure in search of a point but Valladolid went the closest to netting in the final stages, as Oscar Gonzalez knocked Vladet Rama's cutback over the crossbar.
The loss leaves Barcelona a point behind Real Madrid in the title race but Carlo Ancelotti's men welcome Levante to Santiago Bernabeu on Saturday.
Real will begin the day in second, however, after Atletico's win at Celta lifted them above their city rivals due to having a better head-to-head record.
After a goalless and unspectacular first half, the former Barcelona star latched on to Jonny Castro's disastrous backpass and slotted past Yoel with confidence.
Celta were still reeling from that opening goal when Villa netted a second, coolly stroking an inch-perfect finish into the bottom-left corner following Jose Sosa's right-wing cross.
Villarreal's slim chances of securing a UEFA Champions League place took a serious hit in Saturday's 2-0 loss at struggling Granada, keeping them six points behind Athletic Bilbao.
