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 themelves four points adrift of Real Madrid if the pacesetters beat Levante on Sunday.
Madrid will be expected to win that game, but then Barca were expected to win this and the fact they failed will not only give hope to their two Primera Divison title rivals from the capital, Real and Atletico, but also to Manchester City.
City head to the Nou Camp in midweek needing to overturn a 2-0 deficit if they are to progress through to the quarter-finals of the Champions League.
Barca's shock defeat to Valladolid might have been more unstandable if Martino had decided to rest his top stars ahead of that European date, but the Catalan giants went out at the Jose Zorrilla stadium with almost all of their big guns.
The likes of Lionel Messi, Neymar and Xavi all started and the only noticeable absentee was Andres Iniesta, missing for personal reasons.
Despite that though, they could not overcome struggling Valladolid as they crashed to a second successive away defeat, having previously lost 3-1 to Real Sociedad, and one that could seriously hurt their title defence as well as turn the heat up on Martino.
Valladolid gave an early warning as to what might be in store for Barca when Javi Guerra was denied by Victor Valdes when one-on-one in the fifth minute, but the visitors responded with Messi bringing a fine save out of Marino.
Adriano then fired over as Barca looked to be getting into their stride, but it was Valladolid who took a surprise lead in the 17th minute when Rossi slotted home first time after Guerra's blocked shot had fallen kindly for him inside the area.
It was virtually all Barcelona after that as they sought to avoid a shock loss, but try as they might, they could not get back on terms.
Marino denied Messi and Cesc Fabregas in quick succession before the break before he produced a brilliant save to parry another Messi effort at the start of the second half.
Neymar then shot over for the visitors but as the second half wore on Barca found chances increasingly hard to come by as Valladolid held firm for only their fifth league win of the season, and certainly their most unexpected.