Aims: To evaluate the biodegradability of 2,4-DNT using an anaerobic filter (AF) combined with a biological aerated filter (BAF), and elucidate the degradation mechanism of 2,4-DNT and analyze the bacterial community of the reactors over a long period of operation. Methods and Results: The pilot test experienced wide fluctuations influent concentrations and there was lower than 0.50 mg l-1 of 2,4-DNT in the effluent of the system. The removal efficiency was above 99%. GC-MS analysis demonstrated that 2,4-DNT was mainly reduced to 2-amino-4-nitrotoluene (2-A-4-NT), 4-amino-2-nitrotoluene (4-A-2-NT), and 2,4-diaminotoluene (2,4-DAT) during the anaerobic reaction. In addition, ethanol was added into the influent as the electron donor. Because of the use of part ethanol as an auxiliary carbon source, more than twice the theoretical requirement of ethanol was needed to achieve a high 2,4-DNT removal efficiency (> 93%). ESEM observations showed that the carrier could immobilize micro-organisms, which flourished more in reactors operating over longer periods. Further research by PCR-DGGE revealed that new 2,4-DNT-resistant bacterial had been generated during the stress of 2,4-DNT for 150 days. The dominant species for 2,4-DNT degradation were identified by a comparison with gene sequences in GenBank. Conclusions: 2,4-DNT could be effectively degraded by the combined process and ethanol played an important role in the biotransformation. The proposed transformation pathway of 2,4-DNT was concluded. During the 150-day operation, some microbial taxa unaccustomed to 2,4-DNT died out and some new 2,4-DNT-resistant microbial taxa appeared. Significance and Impact of the Study: The study provides a novel method for the bioremediation of 2,4-DNT, which is difficult to degrade by traditional biological methods. The most 2,4-DNT-resistant microbial taxa have not been reported elsewhere and they may be helpful to the treatment of actual 2,4-DNT wastewater.