Here, we describe a strategy to obtain nanoporous liquid-crystalline (LC) membranes by incorporating a photocleavable ortho-nitrobenzyl group in polymerizable columnar liquid crystals. Two derivatives were synthesized with propylene and nonylene spacers, respectively, between the ionic and the photocleavable moieties to introduce various size nanopores after photocleavage. The membranes were prepared by photopolymerization in the LC states, followed by photocleavage and washing with methanol. The resulting membranes show a virus rejection of 99.99%. Although the rejection value remained almost the same for the two membranes, water flux increased with increasing the length of the alkyl spacers. These membranes were found to be almost free from pinhole defects. The present study offers a new methodology for the development of nanoporous membranes with organized nanostructures for separation technologies.