Mammalian sperm undergo several maturation steps after leaving the testis to become competent for fertilization. Important changes occur in sperm within the female reproductive tract, although the molecular mechanisms underlying these processes remain unclear. To investigate sperm membrane remodeling upon sperm maturation, we developed transgenic mouse lines carrying glycosylphosphatidylinositol (GPI)-anchored enhanced green fluorescent protein (EGFP-GPI) and traced the fate of this fluorescent protein during the fertility-acquiring process in sperm in vitro and in vivo. When the GFP-labeled sperm were treated with compounds for promoting the acrosome reaction, EGFP-GPI was released from the sperm surface crosslinked with characteristic relocation of a lipid raft marker ganglioside GM1. Sperm ejaculated into the uterus strongly expressed EGFP-GPI in the head region, whereas a part of the oviductal sperm lost fluorescence in a manner that was dependent on the presence of angiotensin-converting enzyme (ACE). Moreover, sperm on the zona pellucida of eggs in the oviduct were all found to have low levels of GFP. These results suggest that sperm undergoing GPI-anchored protein release associated with reorganization of lipid rafts and the acrosome reaction acquire fertilization potential.