Abstract: Various casein phosphopeptides (CPPs) were extracted by Ca 2+ /ethanol precipitation and ion exchange chromatography through hydrolyzing milk casein enzymaticlly, and then were determined in vitro and in vivo. The results showed that CPPs3 (MW 4 066, N/P 5.48) derived from bienzymatic hydrolysate (trypsin and alcalase) is the best one in calcium binding capacity in vitro and in improving Ca 2+ absorption in mice (Ca 2+ absorption rate is 56%). These two data were superior to the CPPs derived from single enzymatic hydrolysate and other CPPs products.