Mammalian fertilization is a multistep process that culminates in the fusion of the sperm and egg plasma membrane. It is widely accepted that the equatorial segment of the acrosome-reacted sperm is important in initiating fusion with the egg plasma membrane during fertilization. There are various proteins known to be distributed only in the equatorial segment of sperm. The role of these proteins must be clarified to understand the membrane fusion process. We produced a mouse line that lacked SPESP1 (sperm equatorial segment protein 1) and analyzed the fertilizing ability of the sperm. The average number of pups that were fathered by Spesp1(+/-) and Spesp1(-/-) males was significantly lower than that of wild-type fathers. In these mouse lines, fewer sperm were found to migrate into oviducts and fewer eggs were fertilized. The Spesp1(+/-) and Spesp1(-/-) sperm showed a lower fusing ability compared with the wild-type sperm. The disruption of Spesp1 was shown to cause an aberrant distribution of various sperm proteins. Moreover, scanning electron microscopy revealed that the membrane in the equatorial segment area, which usually forms an acrosomal sheath, disappears after acrosome reaction in Spesp1-deficient mice. It was demonstrated that SPESP1 is necessary to produce the fully 'fusion competent' sperm.