In a focused library of glycolipid-based hydrogelators bearing fumaric amide as a trans–cis photoswitching module, several new photoresponsive supramolecular hydrogelators were discovered, the gel–sol/sol–gel transition of which was pseudo-reversibly induced by light. Studying the optimal hydrogel by NMR spectroscopy and various microscopy techniques showed that the trans–cis photoisomerization of the double bond of the fumaric amide unit effectively caused assembly or disassembly of the self-assembled supramolecular fibers to yield the macroscopic hydrogel or the corresponding sol, respectively. The entanglement of the supramolecular fibers produced nanomeshes, the void space of which was roughly evaluated to be 250 nm based on confocal laser scanning microscopy observations of the size-dependent Brownian motion of nanobeads embedded in the supramolecular hydrogel. It was clearly shown that such nanomeshes become a physical obstacle that captures submicro- to micrometer-sized substrates such as beads or bacteria. By exploiting the photoresponsive property of the supramolecular nanomeshes, we succeeded in off/on switching of bacterial movement and rotary motion of bead-tethered F1-ATPase, a biomolecular motor protein, in the supramolecular hydrogel. Furthermore, by using the photolithographic technique, gel–sol photopatterning was successfully conducted to produce sol spots within the gel matrix. The fabricated gel–sol pattern not only allowed regulation of bacterial motility in a limited area, but also off/on switching of F1-ATPase rotary motion at the single-molecule level. These results demonstrated that the photoresponsive supramolecular hydrogel and the resulting nanomeshes may provide unique biomaterials for the spatiotemporal manipulation of various biomolecules and live bacteria.