An experiment on the separation of hydrogen isotopes has been carried out by using a thermal diffusion column with a “cryogenic-wall” cooled by liquid nitrogen. The separation factor was compared with that of a ordinary column cooled by water, and the separation factor for the “cryogenic-wall” column is higher than that for the “water cooled wall” column. Moreover, the separation factor obtained by a 473 K operation of the hot wire in the “cryogenic-wall” system was found to be greater than that by 1073 K operation. Probably because the isotopic exchange reaction between H2 and D2 was suppressed in 473 K operation; there was no HD component observed in this case, while an equilibrium amount of HD component was immediately detected in 1073 K operation.