Worldwidely,substitution of plant chymosin for calf rennet has always been the hot topic of dairy science. Ginger protease with milk coagulating activity was isolated from ginger root. The hydrolysis of casein isolates and casein in skim milk was analyzed by Urea SDS-PAGE,RP-HPLC,and MALDI-TOF / MS. The results showed that the primary hydrolyzates of κ-casein proteolyzed by ginger protease were stable,and didn't undergo further proteolysis. At temperatures higher than 60℃,the main products were two N terminal hydrophobic peptides κ-CN( f 1-90) and κ-CN( f 1-102) with κ-CN( f 1-121) to a lesser amount. While at low temperatures,large amount of peptides with molecular weights higher than that of κ-CN( f 1-121) were formed. Ginger exhibited proteolysis on κ-casein,but didn't show significant hydrolysis on α S1-,α S2-,and β-casein in skim milk system. Proteolysis of κ-casein occurred at Thr121-Ile122,and generated a hydrophobic N terminal peptide κ-CN( f 1-121). The milk coagulating mechanism of ginger protease was demonstrated to be that the κ-casein was hydrolyzed at peptide bond of Thr121-Ile122,which destroyed the stability of casein micelle and formed gels.