Methyl tert-butyl ether (MTBE) is an additive used to oxygenate gasoline to improve air quality by reducing tailpipe emissions of carbon monoxide and ozone precursors. Although several toxicity studies in rats have been conducted to examine the acute, subchronic, and chronic toxicities by employing various routes of exposure to MTBE, few data were available on the effects of MTBE exposure on blood. In this study, MTBE was administered to rats at dose levels of 0, 400, 800, and 1600 mg/kg/day, respectively. After 2- or 4-weeks treatment period, rats were euthanized and blood was collected for the assay of hematological indicators and blood biochemistry indicators. Some organs, including brain, heart, liver, spleen, lung, kidneys, testes, epididymis, thymus, and prostate, were immediately removed and weighed. Possible subchronic health effects of MTBE exposure by gavage were evaluated on mortality, body weight, relative organ weight, hematology, and blood biochemistry indicators in male Sprague-Dawley rats. The results indicated that MTBE did not disrupt the growth rate of rats. Relative organ weight showed change in heart, liver, kidney, testes, thymus, and prostate. In the 2-week treatment, MTBE exerted toxicity on white blood cell count, including lymphocyte, granulocyte, and eosinophil. This finding was especially strong at 1600 mg/kg/day MTBE. In the 4-week treatment, hemoglobin at high dose MTBE significantly increased. The results of the assay for the biochemistry indicators and relative organ weight indicated that MTBE could impair liver and kidney functions and also have adverse effects on lipid metabolism and immune system. It was conducted that subchronic MTBE exposure induced the adverse effects occurring in the relative organ weight, the hematological indicators, and the biochemistry indicators under high MTBE dose.