The move threatens to destabilise one of the most highly-rated teams in the tournament, with La Roja set to play Portugal on Friday.
Julen Lopetegui has been sacked as manager of Spain, after taking the Real Madrid job on Tuesday, reports Goal.
Lopetegui was announced as Zinedine Zidane's successor earlier this week in a move that surprised many.
The announcement initially claimed that the 51-year-old would take the reigns at Santiago Bernabeu after the conclusion of the tournament, but La Roja have acted now.
"We have to decided to fire the national coach," Spanish Football Federation president, Luis Rubiales, said. "What we have achieved in getting here is due in great part to him, and we must thank him and wish him luck. "
Rubiales insists he does not feel undermined, but has suggested that Real acted in a manner unbecoming of their status.
“I don’t feel betrayed. Lopetegui has done impeccable work. How all this has been done is something else.
"I have spoken with the players and I can guarantee they will do everything, along with the new coach, to take the team as far as possible.
"Real Madrid look for the best coach, that is fine by me. I have no opinion about how Real Madrid act. But the federation have an obligation, to represent all Spaniards. "
The stunning development threatens to destabilise a team regarded as one of the favourites to win the tournament in Russia.