A two-dimensional zeolitic imidazolate framework (ZIF), ZIF-L, with unique anisotropic pore system makes it an excellent material for investigating ZIF membranes with crystallographically preferred orientation and their effects on gas separation properties. A b-oriented ZIF-L membrane was successfully prepared from a randomly oriented seed layer. In contrast, a c-oriented ZIF-L crystal layer was attached to the support through the vacuum filtration in the presence of polyethyleneimine. After a short-time secondary growth, the oriented layer grew into a continuous membrane with a high degree of c-out-of-plane orientation. The gas separation studies demonstrated the orientation-dependent separation behavior. In the single gas permeation experiments, the c-oriented ZIF-L membrane has higher ideal selectivities of 8.1 for H2/N2 and 24.3 for H2/CO2 compared to the corresponding values of 3.9 and 5.5 for b-oriented ZIF-L membrane. The results of binary gas permeation showed decreased separation factors of c-oriented membrane, but were still much higher than those of b-oriented membrane. This work provides new insights into controlling ZIF membrane orienation for achieving desirable gas separation performance.