Abstract: The aim of this study was to ascertain the effect of exogenous cathepsin inhibitor (E-64) on the viability of porcine oocytes developed in vitro. The maturation medium was added with 1, 5, 10 μmol/L exogenous cathepsin inhibitors (E-64) respectively, and the porcine cumulus oocyte complexes (COCs) were matured in vitro for 46 hours, the first polar body rate was calculated. Then in vitro fertilization and parthenogenetic activation were conducted, and the cleavage rate and the blastocyst rate were analyzed. The results showed that the first polar body rate, as well as the associated cleavage rate and blastocyst rate in maturation medium added with 1 μmol/L E-64 were significantly higher than that of other three groups. After parthenogenetic activation, there was no significant difference in the cleavage rate among the four groups, while the blastocyst rate in maturation medium added with 10 μmol/L E-64 was significantly lower than that of other three groups. It could be concluded that the suitable supplement of E-64 for in vitro nuclear maturation of porcine oocytes, as well as the cleavage and embryonic development, should be 1 μmol/L.