After addition of Cu2+ into the anodic and/or cathodic chamber, the effect of Cu2+ on the internal resistance and its distribution, power output and coulombic efficiency of dual-chamber microbial fuel cell (MFC) was investigated in this manuscript with the aid of analyzing the distribution of copper speciation. It could provide helpful information for correlative research on treatment of copper-containing wastewater by MFC. It showed that the addition of 10 mg x L(-1) Cu2+ into the anodic chamber inhibited the microbial activity, and increased the anodic activation resistance as well as the apparent internal resistance, consequently reduced the power output and coulombic efficiency of the system. However, the addition of 500 mg x L(-1) Cu2+ into the cathodic chamber significantly reduced the cathodic activation resistance as well as the apparent internal resistance, while improved the power output and the coulombic efficiency. Cu2+ in the anodic chamber was not transfered into the cathodic chamber. When adding Cu2+ into the cathodic chamber, it was mainly reduced and deposited on the cathodic chamber. It could also be transferred/diffused to the anodic chamber across the proton exchange membrane (2.8%) because of its concentration difference, thus affecting the microbial activity and power output. Only a small amount of Cu2+ remained in the supernatant of the cathodic chamber at the end of experiment.