The role of melatonin in protecting spermatozoa from different kinds of injury has been widely reported. The present study aimed to test whether treatment of buffalo bulls with melatonin could ameliorate sperm function during nonbreeding season under tropical condition. Ten Murrah buffalo bulls were randomly allocated into control and treated groups of equal numbers of bulls to study the effect of melatonin on semen characteristics, seminal plasma constituents, blood plasma hormonal levels, and antioxidant enzyme activities during nonbreeding season. Treated bulls were implanted with melatonin (18 mg/50 kg of body weight) for a period of 2 mo. During this period, semen was collected twice a week, and blood samples were collected weekly to determine plasma concentration of melatonin and LH and activities of antioxidant enzymes. During nonbreeding season, melatonin implantation improved semen characteristics by increasing (P < 0.05) percentages of sperm with forward motility, viability, total motile sperm, and rapid motility, average path, curvilinear, and straight-line velocity and amplitude of lateral head displacement and decreasing (P < 0.05) percentages of abnormal sperm and linearity index as compared to the control group. Furthermore, melatonin implantation increased (P < 0.05) seminal plasma concentrations of total protein, albumin, and cholesterol and decreased (P < 0.05) seminal plasma aspartate aminotransferase activity. In addition, melatonin-implanted bulls exhibited an increase (P < 0.05) in red blood cells superoxide dismutase activity compared to untreated bulls. In conclusion, melatonin implantation successfully improved semen quality of buffalo bulls during nonbreeding season under tropical condition.