Voltage reversal in stacked microbial fuel cells (MFCs) is a significant challenge that must be addressed, and the information on its definite cause and occurrence process is still obscure. In this work, we first demonstrated that different anodic reaction rates caused voltage reversal in a stacked MFC. Sluggish reaction rates on the anode in unit 1 of the stacked MFC resulted in a significantly increased anode overpotential of up to 0.132 V, as compared to negligible anode overpotential (0.0247 V) in unit 2. This work clearly verified the process of voltage reversal in the stacked MFC. As the current was gradually increased in the stacked MFC, the voltage in the stacked unit 1 decreased to 0 V prior to that of the stacked unit 2. Then, when the voltage in unit 1 became 0 V, it was converted from a galvanic cell to an electrochemical cell powered by unit 2. We found that the stacked unit 2 provided electrical energy for the stacked unit 1 as a power supply. Finally, the anode potential of the stacked unit 1 significantly increased over cathode potential as current increased further, which caused voltage reversal in unit 1. Voltage reversal occurs in stacked MFCs as a result of non-spontaneous anode overpotential in a unit MFC that has sluggish anode kinetics compared to the other unit MFCs.