Objective： To study the molecular mechanism of epididymal protease inhibitor（Eppin） modulating the liquafication of semen. Methods： Human semenogelin cDNA（nucleotides 82-849） and Eppin cDNA（nucleotides 70-423） were generated by PCR and cloned into pET-100D/TOPO.Recombinant Eppin and Sg were produced by BL21（DE3）. The association of Eppin with Sg was studied by far-western and radioautography.In vitro the digestion of Sg by PSA in the presence or absence of recombinant Eppin was studied. The effect of anti-Q20E（N-terminal） and C-terminal of Eppin on Eppin-Sg binding was monitored. Results： Eppin binds Sg on the surface of human spermatozoa with C-terminal Eppin（aa75-133）.Recombinant Sg was digested with PSA ，many low molecular weight fragments were produced， when Eppin is bound to Sg，then digested by PSA ，producing incomplete digestion and a 14.5-14.8 kDa fragmen. Antibody binding to the N-terminal of Eppin did not affect Sg digestion. Addition of antibodies to the C-terminal of Eppin inhibited the modulating effects of Eppin. Conclusion： Eppin modulates the digestion activity of PSA through binding Sg.The active site locates at C-terminal.