Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. In addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.