One strain of beta-Proteobacteria that could utilize Methyl tert-Butyl Ether (MTBE) as sole carbon and energy resource was used for MTBE degradation in closed system. The culture conditions showed significant effect on the MTBE degradation and the optimum conditions were as following: original pH 7.2, initial cell concentration 10(7) cells/mL and initial MTBE concentration 25 mg/L. The effect of dissolved oxygen (DO) in medium of closed culture system on MTBE degradation was evaluated. It was found that supplication of oxygen into the closed system could significantly promote MTBE degradation. The analysis of intermediate metabolites of MTBE degradation by GC-MS showed that tert-butyl alcohol, isopropanol and acetone were the main intermediate metabolites. The concentration change of metabolites during the whole period of MTBE degradation suggested that the metabolic pathway of MTBE degradation was similar to the "acetone pathway".