Melatonin is an indolamine derived from the amino acid tryptophan, produced in the pineal gland and characterized by its ability to sequester reactive oxygen species (ROS). The aim of this study was to evaluate the effect of melatonin supplementation to Tris-Gema diluent on the cryopreservation of bovine semen. Fifty-four ejaculates from Guzerá bulls were used. The semen was diluted in TRIS-gema, with the addition of melatonin, at the following experimental concentrations: control group - 0miM; Melatonin - 10miM; Melatonin - 100miM; Melatonin - 200miM. They were then filled into 0.25mL straws and cryopreserved. After thawing at 37°C for 30 seconds, the aliquots were analyzed for the Rapid Thermal Resistance Test (RTT), plasma membrane (PM) integrity, acrosome (Ac), mitochondrial membrane potential (MMP) and sperm kinetics. The percentages of motility were higher (p