The objective of this study was to investigate the role of calmodulin-dependent protein kinase II (CaMKII) during fertilization in the pig. Since it has been reported that CaMKII is involved in the capacitation and acrosome reaction of spermatozoa, we tested whether supplementation with the CaMKII inhibitor, KN-93, in the fertilization medium affected sperm penetration. The results showed that the addition of KN-93 in the fertilization medium significantly reduced the rate of sperm penetration into oocytes. However, pre-treatment with KN-93 before in vitro fertilization (IVF) did not significantly affect sperm penetration, but it did affect pronuclear formation in a dose-dependent manner. In the oocytes pre-treated with KN-93 before IVF and then co-cultured with spermatozoa without the drug, the decrease in p34cdc2 kinase and the cyclin B1 level were significantly suppressed as compared with those in penetrated oocytes without treatment with KN-93. However, the decrease in MAP kinase activity was not affected by KN-93. Additional treatment with KN-93 after Ca2+ ionophore treatment also inhibited the reduction in p34cdc2 kinase activity and the cyclin B1 level, but not MAP kinase activity. Treatment with KN-92, an inactive KN-93 analogue, did not significantly affect sperm penetration and pronuclear formation. In conclusion, the activation of CaMKII by artificial stimuli or sperm stimulated the disruption of cyclin B1 and the inactivation of p34cdc2 kinase, but did not affect MAP kinase inactivation during oocyte activation in pigs.