Hun Sen said Monday he is ordering the ban after Buddhist monks told him the activity runs contrary to Buddha's teachings on the sanctity of life and encourages violence.
The practise is also unhygienic, Hun Sen said during a "clean city" forum in the capital.
He said restaurants may still grill and sell spit-roasted beef, but must cook it out of customers' sight. He added that they can advertise it with signs or pictures.
Cows are treated with respect in Buddhist Cambodia, which is influenced by Hindu culture.
The government issued a directive last month banning the practice but it was widely ignored.
