Cryopreservation induces sublethal damage to the spermatozoa, which leads to reduce their fertile life. The objective of the present study was to investigate the effect of melatonin on freezability, ultrastructure and fertilizing potentials of the buffalo spermatozoa. Buffalo spermatozoa were cryopresreved with Tris egg yolk extender containing 7% glycerol supplemented with (0.100, 0.250, 0.500, 0.750 and 1 mM melatonin) or Tris-based extender only (control). Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability and acrosomal integrity, ultrastructure changes and fertilizing potentials. The current results clearly indicated that adding 0.1 mM melatonin to Tris extender significantly improved (P