Processes for separating hydrogen isotopes are important for future energy applications. Several separation methods are based on electrolytic process; however, electrolysis consumes large amounts of electric energy. In this study, we demonstrate deuterium isotope separation from a mixture of H-2 and D-2 gases using a polymer electrolyte fuel cell stack. To identify the most efficient process, we investigated two flow patterns for the fuel gas, namely, parallel and serial flow. The electrical power of the stacks depended on the flow pattern when a high current was generated. We attribute this dependence on membrane dehydration and water droplet formation in the serial flow, which passed through the single cells in a straight path. However, the stack with the serial path showed a high separation factor (alpha = 6.6) indicating enrichment of deuterium water during the operation. The long reaction path of the fuel gas contributed to effective separation. The fuel utilization in individual cells suggested the potential for even more effective separation processes by a serial flow path. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.