Head coach Reinaldo Rueda had named a 24-man squad for Ecuadors international friendly against the Netherlands on Saturday, but he has added a further six players to finalise his initial World Cup side.
Edison Mendez - Ecuadors third-most capped player in their history - John Narvaez, Michael Arroyo, Oswaldo Minda, Angel Mena and Cristian Penilla have been included alongside captain Valencia.
Mendez, 35, has made 108 appearances for the national team, 59 short of record holder Ivan Hurtado.
All nations participating at the World Cup finals are required to submit their official 23-man squads by June 2, though uncapped Stuttgart young-gun Carlos Gruezo is expected to survive the cull.
Cristian Ramirez, Renato Ibarra and Christian Noboa are the only other players from the preliminary selection to be plying their trade in Europe.
Liga MX top scorer Enner Valencia will attempt to fill the void up front, after star striker Christian Benitez, 27, died of a heart attack following a cup game for Qatari outfit El Jaish in July last year.
Ecuador will go head-to-head with the Netherlands, Mexico and England in the lead up to Brazil, before opening their World Cup campaign against Switzerland in Group E action on June 15.
France, the 1998 champions, and Honduras complete the group.
Squad in full:
Goalkeepers: Maximo Banguera (Barcelona, Ecuador), Adrian Bone (El Nacional), Alexander Dominguez (Liga de Quito)
Defenders: Gabriel Achilier (Emelec), Walter Ayovi (Pachuca), Oscar Bagui (Emelec), Frickson Erazo (Flamengo), Jorge Guagua (Emelec), John Narvaez (Emelec), Juan Carlos Paredes (Barcelona, Ecuador), Cristian Ramirez (Fortuna Duesseldorf)
Midfielders: Michael Arroyo (Atlante), Segundo Castillo (Al Hilal), Carlos Gruezo (Stuttgart), Renato Ibarra (Vitesse Arnhem), Fidel Martinez (Tijuana), Edison Mendez (Santa Fe), Oswaldo Minda (Chivas USA), Christian Noboa (Dynamo Moscow), Pedro Quinonez (Emelec), Luis Saritama (Barcelona, Ecuador), Antonio Valencia (Manchester United)
Forwards: Jaime Ayovi (Tijuana), Felipe Caicedo (Al-Jazira), Angel Mena (Emelec), Jefferson Montero (Morelia), Cristian Penilla (Barcelona, Ecuador), Joao Rojas (Cruz Azul), Enner Valencia (Pachuca), Armando Wila (Universidad Catolica)