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We evaluated the role of calpain in human sperm for fertilization. Semen collected manually from healthy donors with informed consent was liquefied and following percoll gradient centrifugation. After exposure to different concentration of progesterone (P) or under hypoxic condition (H), the samples were used for immunostaining, SDS-PAGE and western blot analysis. The increase of calcium ion concentration in the sperm was observed by fluorescent microscope (ARGUS-50CA) system using Fura 2-AM. The role of calpain for fertilization was speculated from the results of Hamster penetration test, Hamilton Motility Analyzer, Triple satin method and Acrobeads test using calpain inhibitors. Immunostaining in human sperm was observed using antibodies against the micro-calpain in the types of whole acrosome, equatorial segment, head segment, neck segment or neck and tail segment. Most of these demonstrated acrosome type staining with anti-pro micro-calpain antibody. Western blot analysis revealed P or H treatment to cause a concentration or time-dependent activation of micro-calpain. In addition, calpain inhibitors significantly reduced the acrosome reaction and penetration. The results suggest that micro-calpain in human sperm plays an important role for the fertilization.