Chivas USA midfielder Oswaldo Minda has been called up by coach Reinaldo Rueda in Castillo's place for the tournament, starting Thursday, reports Xinhua quoting media.
The 32-year-old Castillo, who now serves at Al-Hilal of Saudi Arabia, is a veteran midfielder who played for Ecuador at the 2006 World Cup in Germany. He has failed to recover from a knee injury in a tackle with Mexico's Lius Montes during a friendly in Arlington, Texas May 31.
It was a last minute call after new trials and an MRI revealed that Castle "is not fit" to play in the World Cup by a severe sprain, said the Ecuadorian Football Federation (FEF).
Minda made his international debut for Ecuador in 2008 and was a member of the 2011 Copa America side. Minda, who plays the same defensive midfield role as Castillo, was originally in the 30-man preliminary roster before being dropped by Reinaldo Rueda.
The team travelled on Monday from Quito to Brazilian city Viamao, 30 km from Porto Alegre, where training centre for their participation is located, according to the FEF.
The father of Christian "Chucho" Benitez also accompanied the delegation and wished the team success in the World Cup.
He said he feels the absence of his son, but "we are very confident that Ecuador can spring a surprise, and our selection will give us a joy to all Ecuadorians".
Benitez, Ecuador's deceased top scorer, died suddenly July last year following a heart attack in Qatar.
Ecuador will play their first match June 15 against Switzerland. Ecuador will also face Honduras and France in Group E.