Tight control of sperm capacitation is important for successful fertilization. Glycodelin-S is one of the most abundant glycoproteins in the human seminal plasma. However, its function is unclear. We investigated the role of glycodelin-S on capacitation of human spermatozoa. Binding kinetics experiments demonstrated the presence of two saturable and reversible binding sites of glycodelin-S on human spermatozoa. Differently glycosylated other isoforms of glycodelin, glycodelin-A and -F, did not compete with glycodelin-S for these binding sites, suggesting that the glycodelin-S binding sites are different from those of the other isoforms. Indirect immunofluorescent staining revealed specific binding of glycodelin-S around the sperm head. This immunoreactivity was greatly reduced in spermatozoa that had migrated through the cervical mucus surrogates. Glycodelin-S at physiological concentrations significantly reduced the bovine serum albumin and cyclodextrin-induced cholesterol efflux and down-regulated the adenylyl cyclase/protein kinase A/tyrosine kinase signaling pathway, resulting in suppression of capacitation. Deglycosylation abolished glycodelin-S binding and the effect of glycodelin-S on bovine serum albumin-induced capacitation. This indicates that the carbohydrate moiety of glycodelin-S is critical for the function of the molecule. It is concluded that glycodelin-S in seminal plasma maintains the uncapacitated state of human spermatozoa.