Experimental study for separation of hydrogen isotopes has been performed by using a ‘cryogenic-wall’ thermal diffusion column refrigerated by liquid nitrogen. The column separated H-D system at total reflux and total recycle operational modes. The dependences of the separation factor on the column pressure and hot wire temperature were examined for the total reflux experiments. The optimum pressure observed was 30 kPa at 1273 K. The maximum separation factor at 473 K was larger than that at 1273 K since HD molecules were not produced on the hot wire by the isotope exchange reaction. The separation factor was exponentially proportional to the hot wire temperature. In the total recycle experiments, the separation factor was measured under a variety of flow rates, positions and compositions of the feed stream. The increase in the feed flow rate deteriorated the separation factor appreciably. The position and composition of the feed stream were also major parameters affecting the separation factor.