Real Madrid forward Lucas Vazquez has been called up to the Spain squad to replace Vitolo, who has a knee injury.
Vitolo, who is on loan at Las Palmas ahead of finalising a move to Atletico Madrid in January, suffered minor medial ligament damage during training on Sunday.
"Yesterday morning in training, the player suffered a strain in the medial collateral ligament of his knee," the Royal Spanish Football Federation (RFEF) said in a statement on Monday.
"After consultation with the medical services of his club and those of the RFEF, the player will leave the squad this evening."
Vitolo has established himself as a key figure in Julen Lopetegui's squad, scoring four goals in five appearances in World Cup qualifying.
The 2010 world champions face Italy in their latest Group G match on Saturday before a trip to Liechtenstein next Tuesday.