Flexibility of metal-organic frameworks (MOFs) is the important feature that is distinguishable from other inorganic porous materials such as zeolite and porous silica. Especially, large changes in the unit cell parameters upon exposure of specific adsorbates showing expansion and contraction, called breathing effect, has attracted the attentions in the field of separation and sorption. In-situ dynamic change of pore aperture triggered by external stimuli, especially gas adsorption, is a very useful phenomenon for the separation of mixtures with similar size and shape (e.g., isotope mixture), requiring precise pore size tuning. Herein, for the first time, we report a strategy for effectively separating hydrogen isotopes utilizing the dynamic pore change during the breathing of flexible MOF, MIL-53(Al). The experiment shows that D₂ over H₂ selectivity is strongly related to the state of pore structure of MIL-53(Al). The highest selectivity (S_D2/H2 11.6 at 40 K) was obtained via systematically tuning the pore state of MIL-53(Al) by optimizing the exposure temperature and pressure.