At the anode of a microbial fuel cell (MFC), denitrifying bacteria can coexist with exoelectrogens, enabling an MFC to achieve simultaneous nitrate reduction and electricity generation by the oxidization of organic matter. In this study, an anodic denitrifying MFC (MFC-D) was constructed by incorporating heterotrophic denitriﬁcation. This was compared to a control, MFC without denitriﬁcation (MFC-C). The results showed that MFC-D exhibited higher chemical oxygen demand (COD) removal and power density than MFC-C. Microbial community analysis showed that the proportion of bacteria of the genus Geobacter signiﬁcantly decreased from 72.8% in MFC-C to 29.4% in MFC-D, and that denitriﬁers in MFC-D increased by 36.8% in comparison with those in MFC-C. The COD used for denitriﬁcation was calculated to be 3.7 ± 0.3 g COD/g NO3−-N when MFC-D was fed with different COD/N ratios. The optimal proportion of COD/N in the MFC-D system was 5:1, at which the highest coulombic eﬃciency (CE) of electricity generation and anodic denitriﬁcation were obtained. The maximum voltage output was not inhibited at a low COD/N, but the cycle duration was decreased with decreasing COD/N. Additionally, the bacterial community was more diverse when complex organics were used as an electron donor or in a single-chamber MFC, which subsequently altered the electricity recovery and denitriﬁcation performance. This study provides a new strategy to improve the performance of MFCs in actual applications by the addition of denitriﬁers to the anodic bioﬁlms.