Real Madrid winger Lucas Vazquez has been called into the Spain squad to replace Vitolo, who went down with a knee injury on Sunday.
Vitolo, who is on loan at Las Palmas after a controversial transfer to Atletico Madrid this summer, suffered medial ligament damage during training on Sunday.
The 27-year-old had become a key component to coach Julen Lopetegui's national team setup, scoring four goals in World Cup qualifying thus far.
"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 ahead of two World Cup qualifiers on Monday.
"After consultation with the medical services of his club and those of the RFEF, the player will leave the squad this evening."
The 26-year-old Vazquez has made three appearances for Spain in his career, and is yet to score since receiving his first cap in June of last year.
Spain, who currently sit atop Group G, will take on Italy on Sept. 2 and Liechtenstein on Sept. 5.