The separation of hydrogen isotopes is a critical issue in various fields, such as deuterium or tritium production and the treatment of radioactively contaminated water. In this presentation, we describe the pervaporative separation of hydrogen isotopes using proton conductive membranes and underlying separation mechanism. We investigated the H/D separation factors of perfluorosulfonic acid (Nafion) and polybenzimidazole membranes using pervaporation, and found that both membranes exhibited similar separation factors of approximately 1.026. Water permeation flux through the membranes was highly dependent on their thickness and type, and increased with operation temperature. However, the effect of temperature on H/D separation factor was negligible. We also demonstrated the cascade separation of H/D, indicating the potential application of multi-stage operation. We found that surface transport mechanisms such as hydron hopping contributed the most to H/D separation during the pervaporation process of proton conductive membranes.