Methotrexate (MTX), a chemotherapeutic agent, was shown to adversely affect testis especially seminiferous epithelium. Since melatonin, an endocrine hormone, can normalize testicular functions, its antioxidant property on prevention of MTX-induced testicular damages has never been demonstrated. This study aimed to investigate the protective effect of melatonin on such damage. Sixty adult male rats were divided into 5 groups (n=12/each). Control rats were injected with vehicle whereas MTX, rats were intravenous injected with MTX (75 mg/kg) at days 8 and 15. Melatonin animals were injected with melatonin (8 mg/kg, i.p.) for 15 consecutive days. Rats in preventive or throughout groups were co-treated with melatonin and MTX for 15 or 30 consecutive days. The reproductive parameters including expressions of testicular tyrosine phosphorylation, steroidogenic acute regulatory (StAR), and caspase-3 proteins and malondialdehyde (MDA) level were examined. The results showed that melatonin significantly improved sperm concentration and seminiferous epithelium with decrease of caspase-3expression. Additionally, the intensity of tyrosine phosphorylated proteins of 32 kDa was decreased while 47 kDa was increased in melatonin treated groups compared to MTX group. However, StAR protein expression was not altered compared among groups but testicular MDA levels of melatonin-MTX groups were significantly increased as compared to MTX rats. In conclusion, melatonin improved the MTX-induced testicular damage via antiapoptotic pathway of caspase-3 and increase the tyrosine phosphorylated proteins.