To improve assessment of the acrosome reaction of mouse epididymal sperm, we employed anti-Izumo1 antibody instead of antibodies against acrosomal proteins. The acrosomal states among acrosome-intact, spontaneously acrosome-reacted, truly acrosome-reacted, and probably dead and/or membrane-damaged sperm were clearly distinguished by combined application of anti-Izumo1 antibody, DNA dye Hoechst 33342, and monoclonal antibody MN7 to paraformaldehyde-fixed sperm. When the acrosome reaction of capacitated epididymal sperm on the oocyte zona pellucida was examined using anti-Izumo1 antibody, approximately 20% of sperm bound onto the zona pellucida were acrosome-reacted 30 min after insemination. We also observed the moment of the acrosome reaction of live sperm on the zona pellucida by time-lapse monitoring using fluorescein isothiocyanate-conjugated anti-Izumo1 antibody.