The Manchester United midfielder misses out, while Brazil-born striker Diego Costa gets his first call up to the international squad after a prolific season for Atletico Madrid.
Juventus striker Fernando Llorente has also been left out, with Carles Puyol and David Villa being ignored by Del Bosque.
"This is the squad for the game against Italy and does not mean anything for the World Cup," the Spain coach said at a press conference. "We have a lot of respect for Llorente and really value him, like a lot of other players who didn't make it this time.
"We are not writing off Puyol. We do not have any doubts about him when he's fit and plays regularly."
Costa, meanwhile, previously represented the Selecao at international level, but can switch allegiances as he had yet to play for the Confederations Cup winner in an official game.
Del Bosque has also called up Bayern Munich defensive midfielder Thiago Alcantara for Wednesday's encounter, while his FCB teammate Javi Martinez has also made the cut.
"Costa is in it because of what he has done with Atletico Madrid. Thiago has been doing very well at Bayern. He faces stiff competition in midfield there, but has become a starter."
Squad in full:
Goalkeepers: Casillas (Real Madrid), Reina (Napoli), Valdes (Barcelona)
Defenders: Alba (Barcelona), Albiol (Napoli), Azpilicueta (Chelsea), Juanfran (Atletico Madrid), Martinez (Bayern Munich), Ramos (Real Madrid)
Midfielders: Alonso (Real Madrid), Busquets, Fabregas, Iniesta, Xavi (All Barcelona), Cazorla (Arsenal), Koke (Atletico Madrid), Thiago (Bayern Munich)
Attackers: Costa (Atletico Madrid), Pedro (Barcelona) Navas, Negredo, Silva (All Manchester City)
- Sports & Recreation
- Atletico Madrid
- Diego Costa
- Fernando Llorente
- Carles Puyol
- Bayern Munich