An isotope-selective responsive system based on molecular recognition in porous materials has potential for the storage and purification of isotopic mixtures but is considered unachievable because of the almost identical physicochemical properties of the isotopes. Herein, a unique isotope-responsive breathing transition of the flexible metal-organic framework (MOF), MIL-53(Al), which can selectively recognize and respond to only D₂ molecules through a secondary breathing transition, is reported. This novel phenomenon is examined using <i>in situ</i> neutron diffraction experiments under the same conditions for H₂ and D₂ sorption experiments. This work can guide the development of a novel isotope-selective recognition system and provide opportunities to fabricate flexible MOF systems for energy-efficient purification of the isotopic mixture.