Molecular sieving is a highly efficient method for gas separation because of its ultra-high selectivity. Nevertheless, traditional adsorbents with a molecular sieving effect can only separate a specific gas mixture due to their constant pore apertures. It is still a challenge to continuously fine-tune pore apertures at the sub angstrom scale to separate various gas mixtures in a given porous material. Herein, a temperature-responsive smart molecular gate with a precisely controllable pore size is proposed and validated for molecular recognition and separation, achieved by introducing methoxyl groups into the narrow bottleneck of a metal–organic framework (MOF). The effective aperture size of a smart molecular gate can be continuously tuned from 3.6 to 5.2 Å, covering the size range of commercially important gas molecules. Consequently, the MOF with such a structure exhibits highly selective uptake of several gas mixtures, including N₂/CH₄, CH₄/C₂H₄, C₂H₄/C₃H₆, C₃H₆/C₃H₈, and C₃H₈/i-C₄H₁₀, by controlling the opening degree of the smart molecular gate. Furthermore, to verify its practical application, C₃H₆/C₃H₈ separation performance was systematically evaluated, and excellent selectivity for C₃H₆/C₃H₈ can be achieved at room temperature. This rational design of a smart molecular gate in this work opens a new avenue for the application of smart materials for gas separation.