Oussama Sellami, Lassaad Ouertani and Mohamed Selliti are the three members of Tunisia's provisional squad to miss out on places in the final 23 for the World Cup finals.
While Ouertani and Selliti were fringe players, Sellami's absence is the result of the knee injury he sustained in Thursday's friendly match with Japanese side Oita.
Initially it was hoped that Sellami would only be out for around 10 days. However, further tests have revealed that he has a fracture and he will play no part in the tournament.
Despite the absence of one of their star midfielders, coach Ammar Souyah is still confident that his side can make progress.
Tunisia play Russia, Belgium and Japan in the first phase.
"We studied our opponents on video and also sent our staff to
watch the games between Japan and Norway, and Belgium and France," Souyah said atTunisia's training base in the south western city of Oita.
"Every team is at the same level and each of them has a chance
to advance to the knock-out round."
Tunisia, who are appearing at their third World Cup, play Denmark on 26 May before taking on Russia in their opening match in Kobe on 5 June.
Goalkeepers: Ali Boumnijel (Bastia), Hassen Bejaoui (C.A.Bizertin), Ahmed Jaouachi (U.S.Monastir).
Defenders: Tarek Thabet, Radhi Jaidi, Khaled Badra, Jose Clayton (Espérance), Emir M'kademi, Mohamed Mkacher (E.S.Sahel), Hamdi Marzouki (Club Africain), Raouf Bouzaiene (Genoa), Hatem Trabelsi (Ajax).
Midfielders: Kaies Ghodhbane (E.S.Sahel), Mourad Melki (Espérance), Zoubeir Beya (Besiktas), Hassen Gabsi, Imed Mhedhebi (Genoa), Slim Ben Achour (Martigues), Radh Bouazizi (Borsa).
Strikers: Ali Zitouni, Zied Jaziri (E.S.Sahel), Riadh Jelassi (Club Africain), Adel Sellimi (Freiberg).