For practical implementation of MFC, increasing power generation is important because it is closely related with energy production rate and wastewater treatability. However, it is not known which relative arrangement of anode and cathode gives the best performance, and it is necessary to know electrochemical reference point of the brush anode for this. Five different brush-anode configurations were tested in a single-chambered cubic MFC. By merely changing a brush anode configuration, power and current densities were increased by 20% and 30%, respectively. The horizontally-positioned anode configuration (H1) with the closest anode-cathode distance produced the highest power and current. EIS showed that anode impedance and full-cell impedance were decreased by 60% and 49%, respectively. CE and EE were not significantly affected by the anode-cathode distance, but the horizontal type cells showed relatively higher CE, EE and COD removal rate and shorter batch time. The center of a titanium current collector and the center of carbon fibers of a brush-anode were found to be statistically-significant reference points for MFC electrochemistry. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.