To date, IZUMO1 is the only sperm factor proven to be essential for sperm-egg fusion, and the spermatozoa from Izumo1^-/- male mice completely lose their ability to fuse with egg. It was known that IZUMO1 is hidden under plasma membrane when freshly prepared and migrates onto the plasma membrane after acrosome reaction. However, the dynamics of IZUMO1 during the exposure and its behavior at the moment of sperm–egg fusion were questions to be solved. To visualize the behavior of IZUMO1 in living spermatozoa, we generated a transgenic mouse line expressing mCherry-tagged IZUMO1 (Red-IZUMO1) in a testis specific manner. By using Red-IZUMO1 transgenic mice with the combination of Izumo1^-/- mouse and Green-acrosome transgenic mouse, we examined the precise localization of IZUMO1 before and after acrosome reaction as well as subsequent fertilization processes. IZUMO1 was localized on both inner- and outer-acrosomal membranes in freshly prepared spermatozoa. At the time of acrosome reaction, Red-IZUMO1 demonstrates rapid diffusion onto the whole sperm head and then tends to localize in the medial region of sperm head, called the equatorial segment. The moment of sperm–egg fusion was also imaged live, demonstrating that the diffusion of Red-IZUMO1 onto the egg membrane begins from the equatorial segment, and resultant Red-IZUMO1 showed typical fusion form of distribution. Further experimentation using CD9-GFP egg indicated that such fusion form was given by engulfment of inner-acrosomal membrane into the egg cytoplasm. From this study, we found that the translocation of IZUMO1 at the acrosome reaction occurs from outer-acrosomal membrane to plasma membrane presumably through the formation of membrane-membrane bridges, and that IZUMO1 diffused away from the equatorial segment to egg plasma membrane at the beginning of sperm-egg fusion. Additionally, the endocytotic activity of egg is shown to be prominent immediately after sperm-egg fusion takes place.