Fuel cells (FC) have been developed for automobiles and stationary power units. In addition to a power generator function, we propose a new application of hydrogen isotope separation. In this paper, deuterium (D) separation is investigated by two types of AFCs with platinum (Pt) or ruthenium (Ru) anode catalysts. The characteristics of the AFCs are evaluated by pure protium (H) or deuterium gas separately. In the case of Pt catalyst, the cell current/voltage curves show similar results for both gases. But a remarkable decrease in the voltage value is observed probably due to the mass transportation (diffusion) limitation at Ru catalyst. The limitation effect was larger for D2 than H2 gas. The AC impedance measurements supports the slow reaction rate of D2 gas on Ru catalyst. The separation experiments are verified with hydrogen gas mixed with 1 at% D. The D is diluted in the unreacted gas discharged from AFC with Pt catalyst, but it is concentrated with Ru one. The inverse response may be attributed to the elementary process of the hydrogen oxidation reaction and the difference in the adsorption energy of gas and water molecules on the catalyst surface.