MFI and LTA zeolites were synthesized inside three-dimensionally ordered mesoporous (3DOm) carbon. After the removal of the carbon, a highly crystalline 3DOm-imprinted zeolite with tunable and ordered mesopores was obtained. The nominal mesopore size can be finely controlled from 5 to 15 nm by changing the cage size of the 3DOm carbon. Under different synthesis conditions, the crystal size of the 3DOm-imprinted (3DOm-i) MFI was tuned from approximately 100 nm to several micrometers. The 3DOm-i zeolite was mesoporous and exhibited a spherical shape with a rough external surface. With the synthesis of 3DOm-i zeolites, mixed matrix membranes (MMMs) were prepared by incorporating them into a commercial polyimide matrix (i.e., Matrimid (R) 5218). Both MFI- and LTA-containing MMMs exhibited good adhesion between the zeolites and polymer because of their imprinted mesopore structure and rough external surface. Also, both MMMs exhibited a substantial increase in permeability to all the gas penetrants used in this work (except for NF3), resulting in an excellent N-2/NF3 separation efficiency.