The reproductive-derived serine protease inhibitor Kazal-type (Spink) has been identified in seminal plasma, and Spink–spermatozoa binding has been illustrated in many mammalian species including human. We used mice as experimental animal to study the mode of Spink action in the modulation of mammalian sperm activity. A Spink3-binding zone was cytochemically stained on the sperm head at apical hook separated from intact acrosome, whether the cells were capacitated or not. The Spink3–spermatozoa binding neither changed the population of cells in the uncapacitated, capacitated and acrosome-reacted status nor affected the capacitation-related protein phosphorylation and cell motility enhancement. Despite that, the Spink–spermatozoa interaction resulted in decreasing the intracellular calcium concentration ([Ca2+]i) of the cell head and suppressing both the acrosome reaction induced by Ca+2 ionophore A23187 and the cell fertility. Furthermore, Spink3 seen on the head of spermatozoa in the uterine cavity after coitus could be removed by the trypsin-like activity in the uterine fluid of oestrous females, and free Spink3 in the uterine cavity suppressed the protease activity. We integrated our data to shed light on the molecular mechanism of how Spink and its inhibiting protease are interplayed to modulate the activity of mammalian spermatozoa during their transit in the reproductive tract.