Methyl tert-butyl ether (MTBE) is an oxygenated compound, which has been widely used in Asia, Europe and North America. Although numerous in vitro and in vivo studies have demonstrated the carcinogenicity and the toxicity of MTBE, there is still a lack of data on reproductive system exposure of MTBE in male rodent 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 2 or 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 significant increase in the percentage of abnormal sperm; an irregular and disordered arrangement of the seminiferous epithelium 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 of androgen 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 serum was significantly increased. This finding was especially strong at 1600 mg/kg/day MTBE. In the 2-week treatment, at 1600 mg/kg/day, the mRNA level of 8-oxoguanine DNA glycosidase (OGG1) was significantly decreased, and the mRNA level of the extra-cellular form of superoxide dismutase (SOD(EX)) was significantly increased. Our experiments suggest that relatively high doses of MTBE can exert reproductive system toxicity of male rats and disturb the secretions of T, LH and FSH, possibly due to oxidative stress induced by MTBE.