The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5-7.0 and was inactivated completely by heating at 55℃ for 20 min. The highest level of enzyme activity was obtained at 65 ℃, pH 5.5, in the presence of 25mM CaCl_2. The milk-clotting activity was inhibited only slightly by Na^+ and K^+ and significantly by Cu^(2+), Zn^(2+) and Sn^(2+). The Km value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet.