Separation of highly similar molecules and understanding the underlying mechanism are of paramount theoretical and practical importance, but visualization of the host-guest structure, energy, or dynamism is very difficult and many details have been overlooked. Here, we report a new porous coordination polymer featuring hierarchical porosity and delicate flexibility, in which the three structural isomers of xylene (also similar disubstituted benzene derivatives) can be efficiently separated with an elution sequence inversed with those for conventional mechanisms. More importantly, the separation mechanism is comprehensively and quantitatively visualized by single-crystal X-ray crystallography coupled with multiple computational simulation methods, in which the small apertures not only fit best the smallest para-isomer like molecular sieves, but also show seemingly trivial yet crucial structural alterations to distinguish the meta- and ortho-isomers via a gating mechanism, while the large channels allow fast guest diffusion and enable the structural/energetic effects to be accumulated in the macroscopic level.