Paris Saint-Germain striker Moise Kean was the biggest name to be dropped by Mancini, who kept Kean's PSG teammate Marco Verratti and Inter Milan midfielder Stefano Sensi despite the pair being injured.
The latest squad is not final as it will need to be whittled down to 26 by midnight on Tuesday, a day before Mancini's players are set to meet to prepare for the match against the Czech Republic, which kicks off on June 4.
Compared to the 33-man squad Mancini selected ahead of Friday's 7-0 thumping of San Marino, Sunday's squad list also excluded Cagliari goalkeeper Alessio Cragno, Lazio defender Manuel Lazzari, Vincenzo Grifo, Cristiano Biraghi and Gian Marco Ferrari.
Veteran Juventus defenders Giorgio Chiellini and Leonardo Bonucci were called up, as well as Jorginho and Emerson Palmieri who are fresh from Champions League victory with Chelsea.
Matteo Politano, Matteo Pessina and Atalanta defender Rafael Toloi also get the nod after good displays against San Marino.
The Azzurri are a team reborn under Mancini after their shock failure to qualify for the 2018 World Cup and are undefeated in 26 games.
Italy are in Group A at the Euro and will face Turkey in the tournament's opening match on June 11 in Rome. They will also play Switzerland on June 16 and Wales on June 20, also in the Italian capital.
Italy squad announced on Sunday:
Goalkeepers: Gianluigi Donnarumma (AC Milan), Alex Meret (Napoli), Salvatore Sirigu (Torino)
Defenders: Francesco Acerbi (Lazio), Alessandro Bastoni (Inter Milan), Leonardo Bonucci (Juventus), Giorgio Chiellini (Juventus), Giovanni Di Lorenzo (Napoli), Emerson Palmieri (Chelsea/ENG), Alessandro Florenzi (Paris Saint-Germain/FRA), Gianluca Mancini (Roma), Leonardo Spinazzola (Roma), Rafael Toloi (Atalanta)
Midfielders: Nicolo Barella (Inter Milan), Bryan Cristante (Roma), Jorginho (Chelsea/UK), Manuel Locatelli (Sassuolo), Lorenzo Pellegrini (Roma), Matteo Pessina (Atalanta), Stefano Sensi (Inter Milan), Marco Verratti (Paris Saint-Germain/FRA)
Forwards: Andrea Belotti (Torino), Domenico Berardi (Sassuolo), Federico Bernardeschi (Juventus), Federico Chiesa (Juventus), Ciro Immobile (Lazio), Lorenzo Insigne (Napoli), Matteo Politano (Napoli).