Mammalian seminal plasma is known to contain a decapacitation factor(s) that prevents capacitation and thus, the fertility of sperm. This phenomenon has been observed in experiments conducted in vitro that assessed the inhibition of epididymal sperm fertility by seminal plasma or by the purified decapacitation factor. However, the phenomenon of decapacitation has not yet been characterized in vivo. In the present study, we demonstrate that seminal vesicle protein secretion 2 (SVS2), which is a 40-kDa basic protein and a major component of the copulatory plug, enters the uterus and interacts with ejaculated sperm heads after copulation. The SVS2-binding region of sperm changed from the postacrosomal region to the equatorial segment, while the sperm migrated through the uterus and finally disappeared in the oviduct. Furthermore, SVS2 reduced the fertility of epididymal sperm. The sperm treated with SVS2 decreased the percentage of fertilized oocytes from 60% to 10%. The capacitation state was assessed by protein tyrosine phosphorylation and the comprehensiveness of the acrosome reaction. SVS2 functioned to maintain sperm in the uncapacitated state and to reverse capacitated sperm to the uncapacitated state. We found that the fertility of ejaculated sperm is associated with SVS2 distribution in the female reproductive tract. These results indicate that SVS2 functions as a decapacitation factor for mouse sperm.