Till now, the exact role of melatonin in male infertility hasn’t been fully discovered. Moreover, the intracellular signaling pathways activated by melatonin in buffalo spermatozoa especially on the level of cryopreservation process haven’t been reported. The current study aimed simply to clarify the precise role of this hormone during buffalo semen cryopreservation. Semen samples were obtained randomly from 6 fertile buffalo bulls (aged 3 to 5 years). Weekly, two consecutive ejaculates were collected from each bull for successive six weeks duration using an artificial vagina. The ejaculates were pooled to eliminate variability between the evaluated samples. After that, semen samples were subjected to extension with Tris-based extender supplemented with different concentrations of melatonin (0.1, 0.25, 0.5, 0.75 and 1 mM) Vs. Tris-based extender only (control). Then they were processed to cryopreservation and thawing to assess the different semen characteristics. It had been found that 0.1 mM of melatonin significantly (p