We have studied the effect of spermine-NONOate on acrososme reaction (AR) of buffalo spermatozoa. Heparin capacitated spermatozoa were incubated in presence of 100 µg/mL lysophosphatidyl choline (LPC, T1) or 100 µM spermine-NONOate (T2) or 1 µM 8-Br-cGMP (T3) or 15 µM ODQ (T4) or 100 µM spermine-NONOate+15µM ODQ (T5) in combination to induce AR and assessed by dual staining. AR associated protein tyrosine phosphorylation was detected by immunoblotting and AR was assessed by dual staining. Significant numbers of spermatozoa were acrosome reacted in spermine-NONOate (T2) treated cells as compared to the control (P<0.05). It was also observed that 1.0 µM 8-Br- cGMP caused significantly (P<0.05) higher percentage of AR (32.64±0.37 %) as compared to other 0.1, 0.5, 2.0 µM 8-Br- cGMP. The inhibitor of sGC (ODQ) along with spermine-NONOate (T5) resulted in significantly (p<0.05) higher percentage of AR cells as compared to the ODQ only. Spermine-NONOate caused tyrosine phosphorylation of p20, p38, p45, p69, p80 and p105 proteins. Therefore, this study concluded that Spermine-NONOate is involved in AR of buffalo spermatozoa by phosphorylating the tyrosine proteins though activation of cGMP.