Abstract: A series of hydrogen isotope separation experiment by the twin-bed periodically counter-current flow were carried out using twin combined columns separately packed with Pd and LaNi_4.7Al_0.3, which shows inverse isotope effect. Two kinds of feed gases, the deuterium atomic molar fraction 0.5 and 0.1, were supplied during the effluent curve test and hydrogen isotope separation experiment. The results show that this hydrogen isotope separation by periodically counter-current flow has a quite high separate efficiency. Deuterium atom mole fraction in the product is normally greater than 0.995. For the separation of feed gas that deuterium atomic mole fraction was 0.1 by our improved operation manner, the maximum deuterium atomic mole fraction in product, the maximum enrichment, the deuterium recovery ratio are 0.998, 9.98 and 92.5% respectively, hydrogen atomic mole fraction in exhaust is greater than 0.996, and the full separation coefficient is over 120 000.