The objective of this study was to investigate the effects of salinity on power generation and microbial community structure in the microbial fuel cell (MFC). Three two-chamber MFCs (MFC-A, MFC-B, MFC-C) were used to conduct experiments. The MFC-A was operated sequentially using solutions of 0, 20, 40, 60, and 70 g/L NaCl in the anode chamber of the cell. The MFC-B was operated with solutions of 0 and 40 g/L NaCl and the MFC-C with solutions of 0 and 70 g/L NaCl. Results showed that the salinity inhibited power generation in all the MFCs. In the MFC-A, the maximum voltage outputs and CEs decreased from 660 to 130 mV and from 67% to 4%, respectively, with the NaCl concentrations from 0 to 70 g/L. However, the NaCl concentrations did not affect the removal efficiency of substrate (glucose) in the MFC, which reached 100% at the end of every cycle. Moreover, voltages could be recovered to the maximum value (630 mV) within 60 h after replacing the salt solutions with water. With the solutions of 40 and 70 g/L NaCl in the MFC-B and MFC-C, respectively, no obvious electricity was generated after two cycle operations.