Gerard Pique won the 43rd-minute spot kick that proved the difference between the sides but, following back-to-back draws against Valencia and Athletic Bilbao in the top flight, Ernesto Valverde's men were again short of their fluent best.
Former Blaugrana goalkeeper Jordi Masip impressed in goal for Valladolid, not least after substitute Philippe Coutinho won a second penalty and he plunged to his left to deny Messi.
Referee Juan Martinez Munuera rejected earlier penalty claims from Kevin-Prince Boateng on his first LaLiga start but was persuaded by Pique's slightly theatrical fall under Michel's clumsy challenge.
Masip guessed correctly to his left but was beaten for power and accuracy by Messi, before Pique made himself useful in his own box – getting a vital touch with Enes Unal poised to convert Nacho's cross.
Sergi Guardiola dragged a shot wide as Valladolid continued to acquit themselves well in the second half, although they were indebted to a stunning reaction save from Masip as he denied Messi on the half-volley.
The visiting keeper scrambled from his line effectively to snuff out danger after Boateng sprung a flimsy offside trap in the 55th minute.
Luis Suarez replaced the ex-AC Milan attacker with an hour played and he was soon thwarted by Masip in a one-on-one.
Messi's frustrations approached boiling point as he was booked for protests following a robust challenge from Anuar Mohamed.
He would prove the match-winner, even if Masip did his mood no favours five minutes from time, calmly pouching his headed follow-up from the penalty save.
What does it mean? Barcelona doing enough but nothing more
This was certainly not the performance Valverde will have wanted in the week he signed a surprise contract renewal at Camp Nou. Victory guaranteed Barcelona will return to European action next week holding at least a six-point advantage at LaLiga's summit, with Real Madrid in action against Girona on Sunday.
But the coming months look likely to arrive with some uncomfortable questions. Again Valverde rested key players and the supporting cast failed to convince, while recent displays have more in common with the humbling Champions League exits to Juventus and Roma in the past two seasons than more glorious campaigns.
Masip enjoys Camp Nou return
Never more than a peripheral figure at Barcelona – Saturday's appearance doubled his amount of LaLiga appearances at Camp Nou – La Masia graduate Masip was superb on his return. The early second-half save from Messi was the game's outstanding moment until he denied the great man again from 12 yards. A remarkable stop from Suarez in stoppage time completed a fabulous body of work.
Alena fails to grab opportunity
The academy that reared Masip is more famous for producing playmaking midfielders, although the well has run dry in that regard of late. Carles Alena is the outstanding talent of the most recent crop, a tidy and elegant central operator. But his radar was askew here, with several passes misplaced in clanging fashion. Alena's start came as Ivan Rakitic was rested, but Valverde tellingly summoned the Croatia star in his place 14 minutes from time.
Key Opta facts
- Masip is the goalkeeper with the most penalties saved in the top five European leagues this season (three - level with Cardiff City’s Neil Etheridge).
- Messi is the first player from the top five European leagues to reach 30 goals this season in all competitions.
- Valverde has managed his 410th LaLiga game, becoming 15th in the list of managers with the most games in the competition.
- Barcelona have collected 54 points in 24 games in LaLiga this season, the lowest total by a top side at this stage since 2006-07 (also Barcelona with 49 points).