Gene disruption experiments have proven that the acrosomal protein IZUMO1 is essential for sperm-egg fusion in the mouse. However, despite its predicted function, it is not expressed on the surface of ejaculated spermatozoa. Here, we report the dynamics of diffusion of IZUMO1 from the acrosomal membrane to the sperm surface at the time of the acrosome reaction, visualized using a fluorescent protein tag. IZUMO1 showed a tendency to localize in the equatorial segment of the sperm surface after the acrosome reaction. This region is considered to initiate fusion with the oolemma. The moment of sperm-egg fusion and the dynamic movements of proteins during fusion were also imaged live. Translocation of IZUMO1 during the fertilization process was clarified, and a fundamental mechanism in mammalian fertilization is postulated.