Methyl tert-butyl ether (MTBE) is an oxygenated compound, which has been widely used in Asia, Europeand North America. Although numerous in vitro and in vivo studies have demonstrated the carcinogenicityand the toxicity of MTBE, there is still a lack of data on reproductive system exposure of MTBE in malerodent animals. We studied subacute exposure of MTBE on the reproductive systems of male Sprague–Dawley rats. MTBE was administered to rats at dose levels of 0, 400, 800 and 1600 mg/kg/day. After 2or 4 weeks of treatments, the rats were euthanized, and their serum, epididymis and testes were collected.Significant adverse effects in their reproductive system were observed including: a significantincrease in the percentage of abnormal sperm; an irregular and disordered arrangement of the seminiferousepithelium indicated by a histopathological examination; changed serum levels of testosterone (T),luteinizing hormone (LH) and follicle stimulating hormone (FSH); and decreased levels of mRNA and ofandrogen binding protein (ABP). In the oxidative stress study, results indicated an increased maleic dialdehyde(MDA) content, implying a raised peroxide level, and that the total antioxidant ability in serumwas significantly increased. This finding was especially strong at 1600 mg/kg/day MTBE. In the 2-weektreatment, at 1600 mg/kg/day, the mRNA level of 8-oxoguanine DNA glycosidase (OGG1) was significantlydecreased, and the mRNA level of the extra-cellular form of superoxide dismutase (SODEX) was significantlyincreased. Our experiments suggest that relatively high doses of MTBE can exert reproductivesystem toxicity of male rats and disturb the secretions of T, LH and FSH, possibly due to oxidative stressinduced by MTBE.