© 2019 Hydrogen Energy Publications LLC The separation of hydrogen isotopes has important applications for fundamental science and nuclear engineering. This study investigates isotope separation by stacked polymer electrolyte fuel cells that form part of a combined electrolysis and fuel cell (CEFC) system. Fuel gas containing deuterium (D) was generated by water electrolysis and passed through three fuel cells (FCs) connected in series. Increasing the number of operating FCs in the series greatly improved D separation, but had only a modest impact on power consumption. When all three FCs were individually controlled, the separation efficiency depended on the power condition in each FC. At high current the separation factor of the CEFC system reached over 100 owing to the relationship between fuel gas utilization and separation efficiency.