Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3(-/-)) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3(-/-) mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3(-/-) mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3(-/-) mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3(-/-) mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm-egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm-egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.