A new approach for hydrogen isotope separation using an unsaturated organometallic complex was proposed. Adsorption measurements of [Mn(dppe)2(CO)(N2)](BArF24) (Mn-dppe) (dppe = 1,2-bis(diphenylphosphino)ethane, BArF24 = B[C6H3(3,5-CF3)2]4) using H2 and D2 revealed a significant difference in the adsorption enthalpy of H2/D2 at much higher room temperatures than in previous studies, with D2 molecules being more strongly adsorbed on unsaturated metal sites. Mixed gas adsorption isotherms were calculated at each temperature using IAST, and it was predicted that D2 uptake was much larger than H2 uptake. Column chromatographic separation using the difference in adsorption enthalpy indicated that deuterium could be concentrated, and DFT calculations suggest that this difference in adsorption force is due to the difference in vibrational potentials involved in metal-dihydrogen bonding. This study introduces a new separation approach that could enable hydrogen isotope separation in the ambient temperature range.