Single-chamber microbial fuel cells (MFCs) can efficiently treat wastewater containing nitrate, probably because the interaction between exoelectrogens and denitrifying bacteria may enhance the denitrification activity of MFCs. In this study, the denitrification of nitrate with a wide range of concentrations was investigated by using single-chamber air cathode MFCs. The maximum average denitrification rate of the MFCs inoculated and operated under closed-circuit conditions (Group N-CC) was up to 12.2 ± 0.6 kg NO3--N m-3 d-1 at a high nitrate concentration of 2000 mg NO3-N L-1, which was 74.3% higher than that of the MFCs inoculated and operated under open-circuit conditions and which was significantly higher than those of other MFC systems and many traditional bioreactors. The high denitrification activity of the MFCs of Group N-CC was attributed to the significant reduction of nitrite accumulation through the possible bioelectrochemical nitrite reduction by exoelectrogens that were only enriched at the anodes of the MFCs of Group N-CC. In addition, the MFCs of Group N-CC showed good stability (over 3.5 years) and low apparent activation energy (34.0 kJ mol-1) of the denitrification, indicating the good coexistence of exoelectrogens (Geobacter) and denitrifying bacteria (Thauera) with high performance on denitrification during the long-term operation.