The contamination of methyl tert-butyl ether (MTBE) in underground waters has become a widely concerned problem all over the world. In this study, a novel closed culture system with oxygen supplied by H2O2 was introduced for MTBE aerobic biodegradation. After 7 d, almost all MTBE was degraded by a pure culture, a member of beta-Proteobacteria named as PM1, in a closed system with oxygen supply, while only 40% MTBE was degraded in one without oxygen supply. Dissolved oxygen (DO) levels of the broth in closed systems respectively with and without H2O2 were about 5-6 and 4 mg/L. Higher DO may improve the activity of monooxygemase, which is the key enzyme of metabolic pathway from MTBE to tert-butyl alcohol and finally to CO2, and may result in the increase of the degrading activity of PM1 cell. The purge and trap GC-MS result of the broth in closed systems showed that tert-butyl alcohol, isopronol and acetone were the main intermediate products.