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Worldwide extensive use of plasticized plastics has resulted in phthalates pollution in different environment. Nitrates from industry and agriculture are also widely disseminated in the soils, natural waters and wastewaters. Dimethyl phthalate (DMP) biodegradation by activated sludge cultures under nitrate-reducing conditions was investigated. Under one optimized condition, DMP was biodegraded from 102.20 mg/L to undetectable level in 56 h under anoxic conditions and its reaction fitted well with the first-order kinetics. Using the high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) analysis, mono-methyl phthalate (MMP) and phthalic acid (PA) were detected as the major intermediates of DMP biodegradation. When combined with the determination of chemical oxygen demand (CODCr) removal capacity and pH, DMP was found to be mineralized completely under anoxic conditions. The biodegradation pathway was proposed as DMP → MMP → PA → … → CO2 + H2O.The molar ratio of DMP to nitrate consumed was found to be 9.0:1, which agrees well with the theoretical stoichiometric values of DMP biodegradation by nitrate-reducing bacteria. The results of the non-linear simulation showed that the optimum pH and temperature for the degradation were 7.56 and 31.4℃, respectively.