Acetylene plays a key role in diverse commercial chemicals and high-quality fuel applications, yet its storage poses significant challenges due to its explosive nature. In-situ plasma-assisted CH4 coupling conversion presents a promising alternative for the safe production of C2 chemicals, including acetylene, assuming that efficient downstream separation processes are feasible. This study focuses on the adsorptive separation of acetylene from ethylene to achieve high-purity acetylene with the use of mesoporous metal-organic frameworks (MOFs) as effective selective adsorbents. Herein, we systematically investigate the acetylene/ethylene separation performance of a series of MIL-100 (M = Al, Fe, V, Cr) MOFs. Single-component sorption data first evidenced that MIL-100(Cr) shows the highest affinity to acetylene, supported by Operando Infrared spectroscopy and Density Functional Theory calculations. These analysis revealed the crucial role played by the Cr3+ coordinatively unsaturated sites and the counter-anions (OH-/F-) bound to 1 of the 3 Cr3+ atoms of the oxo-trimer. Grand Canonical Monte Carlo simulations further elucidated the microscopic adsorption mechanisms for each single-component and equimolar binary acetylene/ethylene mixtures. Breakthrough experiments demonstrated that MIL-100(Cr) achieves selectivity ranging from 5 to 23, suggesting its potential for dual high-purity acetylene and ethylene production. Vacuum Pressure Swing Adsorption (VPSA) cycle tests indicated that MIL-100(Cr) achieves high acetylene recovery and moderate purity without a rinse step, while an acetylene rinse step enhances purity for fine chemical raw materials. Overall, this study paves the way toward the promotion of MIL-100(Cr) for future large-scale industrial applications in acetylene harvesting and purification.