Microbial fuel cells (MFCs) can remove and recover metals in wastewater; however, there are relatively few studies of metal removal from soil by MFCs. In this study, we developed a three-chamber soil MFC consisting of an anode, contaminated soil, and cathode chamber to remove heavy metals from soil. The performance of the soil MFC was investigated by assessing the relationships among current, voltage, and Cu migration, and reduction. The developed soil MFC successfully reduced and removed Cu, and the Cu removal efficiency in the cathode surpassed 90% after only 7 days of operation. External resistance had a remarkable effect on the performance of the soil MFC which was depended on cathodic polarization. The pH in the cathode also depended on the external resistance. Lower external resistance were associated with lower pH values, higher Cu removal efficiencies, and greater amounts removed in the cathode. Based on sequential fractionation, the acid-extractable and reducible fractions were the main fractions that migrated within the three-chamber soil MFC. Enhancing the voltage output in the three-chamber soil MFC by increasing the external resistance promoted Cu migration, enriched Cu near the cathode, and facilitated Cu removal. Therefore, the developed three-chamber soil MFC not only supports heavy metal migration from soil towards the cathode, but can also realize reduction of heavy metals in the cathode by adjusting the current or voltage generated by the soil MFC.