<p>Controlling the interlayer stacking of two-dimensional metal-organic frameworks (2D MOFs) is crucial and challenging for optimising their mass transfer and separation performance. Herein, we proposed a V-shaped modulator strategy to control the stacking modes of 2D Zr-BTB by employing three metatopic dicarboxylic acid (CPA = 3,5-di(4-carboxylphenyl)aniline, CPB = 1,3-di(4-carboxylphenyl)benzene, and CPP = 2,6-di(4-carboxylphenyl)-4-(phenyl)pyridine) modulators with distinct terminal functional groups. Quantitative high-angle annular dark-field images analysis revealed that the amino-functionalized modulator (CPA) induced a high proportion of untwisted stacking (≈ 64.6%), whereas the phenyl-terminated modulators (CPB and CPP) predominantly resulted in twisted stacking, with untwisted fractions of only 32.0% and 20.6%, respectively. Density functional theory calculations and independent gradient model analysis indicated that strongly polar–NH₂ interactions of CPA effectively anchored adjacent layers, suppressed interlayer rotational freedom, and promoted untwisted stacking. Furthermore, the untwisted Zr-BTB-CPA as a stationary phase exhibited efficient separation performance for all C₇ to C₁₀ alkane isomers and benzene derivative isomers with complete separation (<i>R</i> values &gt; 1.5). In contrast, twisted stacking of Zr-BTB-CPB and Zr-BTB-CPP showed poor separation performance because of their restricted diffusion. Thermodynamic and kinetic analyses further indicated that the ordered channels in Zr-BTB-CPA enable both effective thermodynamic discrimination and rapid mass transfer. This work introduced a strategy for regulating 2D MOF stacking through rational modulator functionalization, achieving efficient gas chromatographic separation.</p>