The 12-time Grand Slam winner's announcement comes just days after the departure of head coach Andre Agassi from his team.
"After Miami Novak Djokovic and his tennis coach Radek Stepanek decided to end their cooperation,' read a statement on Djokovic's official website.
"The private relationship with Stepanek was and will remain great and Novak has enjoyed working with him and learning from him. He remains grateful and appreciative of all the support he has received from Radek during the last period."
The shake-up in the Djokovic camp comes as the Serbian is struggling to recapture his top form since being sidelined by an elbow injury.
Djokovic is due to return to action at the Monte Carlo Masters later this month.