Recently, interest in single cell analysis has increased because of its potential for improving our understanding of cellular processes. Single cell operation and attachment is indispensable to realize this task. In this paper, we employed a simple and direct method for single-cell attachment and culture in a closed microchannel. The microchannel surface was modified by applying a nonbiofouling polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, and a nitrobenzyl photocleavable linker. Using ultraviolet (UV) light irradiation, the MPC polymer was selectively removed by a photochemical reaction that adjusted the cell adherence inside the microchannel. To obtain the desired single endothelial cell patterning in the microchannel, cell-adhesive regions were controlled by use of round photomasks with diameters of 10, 20, 30, or 50 μm. Single-cell adherence patterns were formed after 12 h of incubation, only when 20 and 30 μm photomasks were used, and the proportions of adherent and nonadherent cells among the entire UVilluminated areas were 21.3%±0.3% and 7.9%±0.3%, respectively. The frequency of single-cell adherence in the case of the 20 μm photomask was 2.7 times greater than that in the case of the 30 μm photomask. We found that the 20 μm photomask was optimal for the formation of single-cell adherence patterns in the microchannel. This technique can be a powerful tool for analyzing environmental factors like cell-surface and cell-extracellular matrix contact.