Glutinous rice wine mash liquor is a traditional food of south of China and its ability to coagulate the milk has been proved. The aim of this work was to extract milk-clotting enzyme from glutinous rice wine mash liquor. A partial purified extract of enzyme was obtained by fractional precipitation with (NH4)2SO4. The fractions obtained by precipitation, 40-90% possessed the milk-clotting activity (MCA) (145.72 U/mg). The 40-90% (NH4)2SO4 fraction was further purified by sephadex G-100 and DEAE-sephadex A-50 with MCA (4,360±50 U/mg), which was confirmed by SDS-PAGE that showed only one band with a molecular mass of 36.0 kDa. Highest MCA was attained at 36 ℃. The enzyme was completely inactivated by heating for 20 min at 60 ℃. The MCA increased with the decreasing of milk pH from 8.0 to 5.5, and it was active at the wide range of pH 1 to 7. The metal ions Mg^(2+), Ca^(2+), Ba^(2+), Mn^(2+), Al^(3+), Fe^(2+) had a very clear function to accelerate milk coagulation whereas Na+ and K+ decelerated the activity slightly. The curd effect of the milk-clotting enzyme has primarily been studied.