Genoa goalkeeper Matia Perrin and Roma striker Mattia Destro both return for the first time since the 2012 friendlies against England and France respectively.
Perrin comes in as number two behind Juventus veteran Gianluigi Buffon due to the poor form of Lazio's Federico Marchetti, while on-form Destro joins a forward line devoid of injury-hit Milan striker Mario Balotelli.
Up and coming Milan defender Mattia De Sciglio is another notable return to the squad having last played for Italy at the Confederations Cup in Brazil.
Paris Saint Germain midfielder Marco Verratti also returns to Prandelli's squad for the first time since a friendly against Argentina in August 2013.
Roma midfielder De Rossi, meanwhile, pays the price for a controversial punching incident during Saturday's scoreless draw at home to Inter Milan.
De Rossi escaped punishment for punching Inter's Argentinian striker Mauro Icardi during a corner kick.
However he has faced trial by television since then and was widely expected to miss out.
It is not the first time the tough-tackling 30-year-old has missed out on selection for the Azzurri due to off the ball incidents.
Defenders: Ignazio Abate (AC Milan), Davide Astori (Cagliari), Andrea Barzagli (Juventus), Leonardo Bonucci (Juventus), Giorgio Chiellini (Juventus), Domenico Criscito (Zenit St. Petersburg/RUS), Mattia De Sciglio (AC Milan), Christian Maggio (Napoli), Gabriel Paletta (Parma)
Midfielders: Antonio Candreva (Lazio), Emanuele Giaccherini (Sunderland/ENG), Claudio Marchisio (Juventus), Riccardo Montolivo (AC Milan), Thiago Motta (Paris Saint-Germain/FRA), Marco Parolo (Parma), Andrea Pirlo (Juventus), Marco Verratti (Paris Saint Germain/FRA)
Strikers: Alessio Cerci (Torino), Mattia Destro (AS Roma), Alberto Gilardino (Genoa), Ciro Immobile (Torino), Lorenzo Insigne (Napoli), Pablo Daniel Osvaldo (Juventus)