The stacking between nanosheets is an intriguing and inevitable phenomenon in the chemistry of nano-interfaces. Two-dimensional metal-organic framework nanosheets are an emerging type of nanosheets with ultrathin and porous features, which have high potential in separation applications. Here, the stacking between single-layer metal-organic framework nanosheets is revealed to show three representative conformations with tilted angles of 8°, 14°, and 30° for Zr-1, 3, 5-(4-carboxylphenyl)-benzene framework as an example. Efficient untwisted stacking strategy by simple heating is proposed. A detailed structural analysis of stacking modes reveals the creation of highly ordered sub-nanometer micropores in the interspacing of untwisted nano-layers, yielding a high-resolution separator for the pair of para-/meta-isomers over the twisted counterparts and commercial HP-5MS and VF-WAXMS columns. This general method is proven by additional nanosheet examples and supported by Grand Canonical Monte Carlo simulation. This finding will provide a synthetic route in the rational design of functionalities in two-dimensional metal-organic framework nanosheet.