Petr Čech was heroic but Gerard Pique’s late winner saw Spain break Czech hearts in Toulouse.
The match was the second fixture of Group D, with Croatia having beaten Turkey yesterday.
There were seven centurions on the pitch, as David Silva was making his 100th appearance for La Roja, while Andres Iniesta, Sergio Ramos, Cesc Fabregas, Čech, Tomáš Rosický and Jaroslav Plašil had already crossed that barrier.
It was one of the least experienced players on the pitch who threatened to break the deadlock, as Čech denied Alvaro Morata on 16 minutes.
The Juventus striker went close again with half an hour played, but the Czech goalkeeper was once again at full stretch to tip his effort round the post.
It was quickly turning into Čech versus Spain, and the Arsenal man made further saves from Silva and Jordi Alba.
The Czechs managed just 32 per cent of possession in the first half, but could have taken the lead on the stroke of half-time.
Having broken quickly, Tomáš Necid turned on the edge of the box, but though his strike was powerful, it went straight at David De Gea.
It was a similar story after the break, and Čech was relieved to see Roman Hubník’s deflected clearance go just wide of his post.
Spain were dominating, but the best chance of the game up to that point fell to the Czech Republic.
Ladislav Krejčí’s free-kick evaded the Spanish defence, but unfortunately from a Czech point of view it was Hubník on the end of it, and the Viktoria Plzen centre-back could only stab weakly at De Gea.
That seemed to inject new life into the Czech Republic, and they almost scored again with 65 minutes on the clock.
Krejčí’s deep cross found Gabre Selassie at the back post, and his header across goal was falling for Pavel Kadeřábek, but Fabregas intervened on the line to prevent the full-back tapping it in.
At the other end, Hubník was equally decisive, nicking the ball from Alba after the full-back had rounded Cech.
With a little over 10 minutes to play, Thiago Alcantara looked certain to break the deadlock, but Krejčí got all the way back into his own box to perform a terrific saving tackle.
The Czech Republic had defended heroically, but it was all for nothing when Gerard Pique came up with a late winner.
Andres Iniesta curled in a superb cross, and his Barcelona teammate was on hand to finally beat Cech.
In injury time, Vladimír Darida had a snapshot, but the ball was beaten away by De Gea.
Spain 1-0 Czech Republic
Pique 88 (S)
Spain: De Gea; Juanfran, Pique, Ramos, Alba; Fabregas (Alcantara, 70), Busquets, Iniesta; Silva, Morata (Aduriz, 62), Nolito (Pedro, 82)
Czech Republic: Čech; Kadeřábek, Sivok, Hubník, Limberský; Darida, Plašil; Gabre Selassie (Šural, 85), Rosický (Pavelka, 88), Krejčí; Necid (Lafata, 75)
Referee: Marcinak [POL]