This study was designed to investigate the effect of L-lysine on post-thaw quality of Nili-Ravi buffalo bull semen. Twenty eight ejaculates were collected from four Nili-Ravi buffalo bulls. Semen samples were pooled and split into five equal aliquots and diluted in Tris-citric acid extender containing different concentrations of L-lysine (0.25, 0.5, 1 and 2mM). As a control, semen was extended without addition of L-lysine. The semen samples were cryo-preserved in liquid nitrogen. Post-thaw semen assessment revealed that sperm motility, viability, plasma membrane integrity, acrosomal integrity, mitochondrial integrity and DNA integrity were significantly improved (P ≤ 0.05) in 2mM L-lysine supplemented group as compared to control. For other groups supplemented with L-lysine; post-thaw motility, acrosomal integrity and mitochondrial integrity were also improved (P ≤ 0.05) for 1mM treated group as compared to control. DNA integrity was improved significantly by all L-lysine treated groups as compared to control. Malondialdehyde concentration was higher (P ≤ 0.05) in control group as compared to L-lysine treated groups. Significantly higher rate of catalase was recorded for 1mM and 2mM L-lysine treated groups as compared to control. In conclusion, the supplementation of 2mM L-lysine in semen extender can be used for cryopreservation of Nili-Ravi buffalo bull semen.