<p>Hierarchical porous metal-organic frameworks (MOFs) integrating micro- and mesopores hold promise for advanced separations but often suffer from understudied negative pore synergy, where interconnected pores compromise selectivity and diffusion. Using the zirconium-based porous coordination network (PCN) PCN-608 as a model, we identify that rapid analyte translocation between meso-hexagonal and micro-triangular channels induces chaotic diffusion, undermining separation efficiency. A channel-isolation strategy is then developed via solvent-assisted installation of barrier ligands (BDC/NH₂BDC) at interconnecting windows. PCN-608-BDC with isolated pores exhibit 8-13 times higher diffusion coefficients for xylene isomers than the pristine PCN-608 monitored by inverse gas chromatography (IGC). Molecular dynamics simulations confirm the restriction of cross-pore migration and the acceleration of diffusion kinetics in PCN-608-BDC. The PCN-608-BDC shows obviously better performance than the pristine material as GC stationary phases and breakthrough adsorbents in separation xylene isomers. All PCN-608 series with a micro-mesoporous mixed structure exhibit excellent xylene uptake. Similar improvements in separation performance are also observed for NU-1000 with isolated pores, validating the universality of the phenomena. By balancing pore connectivity and active site availability, this work establishes channel isolation as a generalizable design principle for optimizing hierarchical MOFs to eliminate the negative pore synergy, offering simultaneous enhancements in selectivity, and stability for gas separations.</p>