Cystatin C (CST3), a cysteine protease inhibitor, is highly expressed in human male accessory glands, thus being a component of seminal plasma in which it has been demonstrated to be associated with prostasomes. Prostasomes have been proposed to regulate sperm capacitation. Also, CST3 was found to be downregulated in semen with oligospermia accompanied by abnormal sperm morphology. Based on this information, we thus evaluated the effect of CST3 on human sperm capacitation in vitro. Sperm were isolated from semen, which conformed to World Health Organization criteria, using PureSperm density gradient centrifugation, and were incubated with varying doses of CST3 under capacitating conditions. Immunocytochemistry results showed that CST3 could bind to human sperm primarily on the postacrosomal region, midpiece, and principal tail. Preincubation of sperm with CST3 suppressed sperm capacitation by blocking upstream signals of capacitation that is the cholesterol efflux from sperm plasma membranes, extracellular calcium ion influx into sperm, and increases in intracellular cyclic AMP. Moreover, the downstream signal transduction of capacitation that is the increases in sperm protein phosphotyrosine content was also inhibited by CST3. The suppressive effect of CST3 on sperm capacitation was further supported by inhibition of acrosome reaction of capacitated sperm. These findings suggest that CST3 may function to prevent precocious capacitation and acrosome reaction and thus preserve sperm fertilizing ability until sperm reach the fallopian tube.