Senior internationals Isco and Thiago Alcantara are the notable inclusions in Julen Lopetegui's squad for next month's tournament.
The decision to include Malaga attacking midfielder Isco and Barcelona midfielder Thiago means they are now extremely unlikely to be included in Vicente del Bosque's senior squad for the Confederations Cup in Brazil later in June.
Spain U21s will be the defending champions and have also included Atletico Madrid goalkeeper Joel Robles - who has impressed on loan at Wigan - plus Bayer Leverkusen right-back Daniel Carvajal and Barcelona striker Cristian Tello in their 23-man squad.
There is no place for Barcelona starlet Gerard Deulofeu, which suggests he may travel to Turkey for the Under-20 World Cup later in June instead.
La Rojita have been drawn in the same group as Russia, Germany and Holland in the tournament which runs from June 5 to 18.
Spain Under-21 squad:
Goalkeepers: David De Gea (Manchester United), Joel Robles (Atletico Madrid), Diego Marino (Villarreal).
Defenders: Martin Montoya (Barcelona), Marc Bartra (Barcelona), Marc Muniesa (Barcelona), Daniel Carvajal (Bayer Leverkusen), Nacho Fernandez (Real Madrid), Alberto Moreno (Sevilla), Inigo Martinez (Real Sociedad), Alvaro Gonzalez (Real Zaragoza).
Midfielders: Iker Muniain (Athletic Bilbao), Koke (Atletico Madrid), Thiago Alcantara (Barcelona), Pablo Sarabia (Getafe), Ignacio Camacho (Malaga), Isco (Malaga), Asier Illarramendi (Real Sociedad), Sergio Canales (Valencia).
Forwards: Rodrigo (Benfica), Cristian Tello (Barcelona), Alvaro Vazquez (Getafe), Alvaro Morata (Real Madrid).