We report an ion-exchanged zeolite as an excellent candidate for large-scale application in hydrogen isotope separation. Ag(I)-exchanged zeolite Y has been synthesized through a standard ion-exchange procedure. The D2/H2 separation performance has been systematically investigated via thermal desorption spectroscopy (TDS). Undercoordinated Ag+ in zeolite AgY acts as a strong adsorption site and adorbs preferentially the heavier isotopologue even above liquid nitrogen temperature. The highest D2/H2 selectivity of 10 is found at an exposure temperature of 90 K. Furthermore, the high Al content of the zeolite structure leads to a high density of Ag sites, resulting in a high gas uptake. In the framework, approximately one-third of the total physisorbed hydrogen isotopes are adsorbed on the Ag sites, corresponding to 3 mmol/g. A density functional theory (DFT) calculation reveals that the isotopologue-selective adsorption of hydrogen at Ag sites contributes to the outstanding hydrogen isotope separation, which has been directly observed through cryogenic thermal desorption spectroscopy. The overall performance of zeolite AgY, showing good selectivity combined with high gas uptake, is very promising for future technical applications.