After finishing in the top six in each of the previous five seasons, Villarreal is in 17th place on the table and in the midst of a relegation battle.
The 'Yellow Submarine' has won just one of its nine league games since September and Calleja has paid the price.
The 40 year-old played for Villarreal before joining the coaching staff seven seasons ago, initially in a role with one of the youth teams.
Calleja won three consecutive titles with the under-19s and stepped up to a position with the senior side in September 2017.
Villarreal finished fifth last year to qualify for the Europa League group stages, and it sits top of Group G in that competition having drawn four of its five games.