Water electrolysis is an efficient method to enrich heavy hydrogen isotopes (tritium and deuterium) in the aqueous phase. Although an alkaline water electrolyzer has been commercialized for mass production of hydrogen, such a method requires additional purification steps to remove electrolytes from the final concentrates. On the other hand, proton exchange membrane water electrolysis (PEMWE) does not require additional electrolyte treatment steps, and PEMWE is operated at higher current density compared to the alkaline water electrolysis. In this study, we investigated deuterium and tritium separation from light water by PEMWE. Separation behaviors at the anode and cathode were analyzed, and H/D and H/T separation factors were compared.