A low molecular mass, naturally occurring acrosin inhibitor has been identified and purified (490-7-fold) from human semen, and kinetic studies have been performed on the association characteristics as well as for the determination of affinity constants (K_i values). The results show thatK_i value (3.34 × 10⁻²) of the inhibitor towards human acrosin is almost three times lower than that of pancreatic trypsin, indicating a much higher specificity and inhibitory property for acrosin. The purified human seminal acrosin inhibitor has a molecular mass of 5.5 kDa and shows a single band using 10–20% gradient SDS PAGE. The work is of great significance for the development of more specific, nontoxic and irreversible inhibitors for human acrosin.