Ribonuclease 9 is a member of ribonuclease A superfamily. It is expressed only in the epididymis. However it lacks ribonucleolytic activity and its function (s) is unknown. Immunofluorescence experiments showed that the location of RNase9 protein on sperm surfaces changed during capacitation and acrosome reaction and the protein migrated from neck to acrosome cap. CTC staining was used to assess the role of RNase 9 on capacitation and acrosome reaction. Compared with the control (PBS with 3%BSA), the spontaneous acrosome reaction induced by progesterone could be suppressed by RNase 9 protein and the AR rate is 6.5 ± 1.2%. The exposure of spermatozoa to RNase9 protein also produced a significant decrease in the intracellular cAMP levels during acrosome reaction with respect to control (111 ± 24.5 versus 187 ± 18 fmol/106 spermatozoa; P<0.05). These experiments confirmed that RNase 9 protein significantly impaired the sperm functions, including capacitation and acrosome reaction, and provide the proof for its potential in male reproductive toxicity.