Water electrolysis is essential for producing hydrogen as an energy source for society. The development of polymer electrolyte membrane water electrolysis (PEMWE) has also raised the potential for new applications of electrolysis technologies. In this study, we demonstrate deuterium (D) isotope separation where pure water containing heavy water is supplied to the anode of a PEMWE. The D separation was evaluated by measuring the concentration in the drained water and in the generated hydrogen gas. The D separation was measured at several of stoichiometry numbers of feed water, λ, which was ratio of the theoretical water consumption to water feed. Notably, at the same stoichiometry, the D separation depended on the volume of the water feed to the anode of the cell and current density. This effect might be explained by complex water transport behavior governed by electro-osmotic flow and back diffusion across the concentration gradient.