The aim of the present investigation was to evaluate the impact of melatonin at different concentrations (1 mM and 2 mM) on the seminal parameters before and after cryopreservation of extended Murrah bull semen. Thirty-six ejaculates, collected with the aid of an artificial vagina twice a week from six Murrah bulls were included in the study. Each ejaculate was diluted in a Tris-citric-acid-fructose-egg-yolk-glycerol (TFYG) extender keeping 80 million sperm per ml and split into three equal aliquots, which were supplemented with melatonin (0, 1 and 2 mM; Treatment C, T1 and T2, respectively), filled in 0.25 mL straws and equilibrated for 4 hr at 4 °C, and then frozen in LN2 vapour. Frozen straws were thawed at 37°C for 30 seconds in a water bath for the post-thaw evaluation. Sperm motility, percent live sperm, sperm abnormalities, in-vitro fertility tests (hypo-osmotic swelling test & cervical mucus penetration distance) and enzyme leakage (AST, ALT) were evaluated at both post-dilution and post-thaw stages. The results revealed that the addition of melatonin, at both 1 mM and 2 mM concentrations significantly (P<0.05) improved sperm motility, sperm livability, and HOS-positive spermatozoa. Further, the sperm abnormalities and enzyme leakage were significantly (P<0.05) lower in melatonin-treated groups than in control group. The results were the best with 1 mM methionine supplementation. In conclusion, the study showed that melatonin at a concentration of 1 mM exhibited superior protection of sperm structures and functions as compared to 2 mM melatonin than the control group.