This study investigated the ameliorative role of melatonin (MLT) and the effects of a long-term intake of high-fructose corn syrup (HFCS) on the male reproductive system. Thirty-six male Sprague Dawley rats were randomly divided into 3 groups as follows: Control, HFCS and HFCS + MLT. Testis and epididymal weights were measured. Malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities, total testosterone levels, testicular histopathological damage scores were evaluated, and immunohistochemical analyses were performed on testicular tissue. Epididymal weights were significantly lower in the HFCS + MLT group than those of the control and HFCS groups. MDA was significantly increased, while SOD and CAT activities were reduced in the HFCS group compared with the control group. Administration of melatonin significantly increased SOD and CAT activities in the HFCS + MLT group. Histopathological evaluation revealed slight hyperaemia and oedema in the stromal tissue of rat testes in the HFCS group. Sperm count and Johnsen's testicular biopsy score (JTBS) were significantly decreased in the HFCS group. Immunohistochemical analysis revealed that HSP, iNOS, MDA, OPN and VEGF values were significantly increased in the HFCS group. However, melatonin ameliorated the immunohistochemical scoring. Our results showed that a long-term intake of HFCS caused testicular damage. Melatonin may be a promising pharmacological agent against testicular toxicity induced by HFCS.