The purpose of this study was to investigate the exogenous melatonin feeding administration to the first puberty stage in male broodstock of the Walking catfish, Clarias macrocephalus. The melatonin level of 0 (Control), 50 (Mt0.05) and 250 (Mt0.25) mg/kg in the diet mixed in isonitrogenous and isocaloric of 37% crude protein and 9.3% crude lipid was applied. The male maturation analysis for this study comprised of gonad histology, testosterone assay, gonadosomatic index, sperm abnormality, live sperm rate, sperm concentration, and sperm kinetic parameters. Significant differences were found in maturation analysis (P<0.05) compared with the control treatment. In addition, the histological analysis found that the mature spermatozoa cells were highest in the melatonin-treated male catfish. The present results showed that exogenous melatonin is able to enhance the reproductive system of male C. macrocephalus. The suitable exogenous melatonin level to enhance the Clarias macrocephalus male broodstock first puberty is Mt0.25 (50 mg/kg melatonin in the diet).