The separation of C₂H₂/CO₂ is not only industrially important for acetylene purification but also scientifically challenging owing to their high similarities in physical properties and molecular sizes. Ultramicroporous metal-organic frameworks (MOFs) can exhibit a pore confinement effect to differentiate gas molecules of similar size. Herein, we report the fine-tuning of pore sizes in sub-nanometer scale on a series of isoreticular MOFs that can realize highly efficient C₂H₂/CO₂ separation. The subtle structural differences lead to remarkable adsorption performances enhancement. Among four MOF analogs, by integrating appropriate pore size and specific binding sites, [Cu(dps)₂(SiF₆)] (SIFSIX-dps-Cu, SIFSIX = SiF₆^2-, dps = 4.4’-dipyridylsulfide, also termed as NCU-100) exhibits the highest C₂H₂ uptake capacity and C₂H₂/CO₂ selectivity. At room temperature, the pore space of SIFSIX-dps-Cu significantly inhibits CO₂ molecules but takes up a large amount of C₂H₂ (4.57 mmol g⁻¹), resulting in a high IAST selectivity of 1787 for C₂H₂/CO₂ separation. The multiple host-guest interactions for C₂H₂ in both inter- and intralayer cavities are further revealed by dispersion-corrected density functional theory and grand canonical Monte Carlo simulations. Dynamic breakthrough experiments show a clean C₂H₂/CO₂ separation with a high C₂H₂ working capacity of 2.48 mmol g⁻¹.