Rational structural design and engineering of MOF membranes is promising for energy-efficient separation. Preferred orientation manipulation delegates an effective approach to improve their separation performance, while room-temperature (RT) synthetic protocol is beneficial to foster their industrial batch production. Nevertheless, it remains challenging to fabricate highly oriented MOF membranes at RT; simultaneously, the density of defective sites in the framework, which is assumed to exert significant influence on the separation performance relying on high affinity-interactions between guest molecules and coordinatively-unsaturated open metal sites, should be deliberately tailored. In this study, we achieve RT synthesis of highly (111)-oriented UiO-66 membrane exhibiting high defect density in the framework through employing Zr6O₄(OH)₄(OAc)₁₂ clusters as zirconium source. Prepared membrane manifests an ideal CO₂/N₂ selectivity of 46.2, which represents the highest value among pure MOF membranes tested in comparable operating conditions. This protocol is expected be expanded for facile and mild preparation of state-of-the-art MOF membranes.