The spatial control and patterning of mammalian cells were achieved by using the universal adhesive property of mussel-inspired polydopamine (PDA). The self-polymerization of dopamine, a small molecule inspired by the DOPA motif of mussel foot proteins, resulted in the formation of a PDA adlayer when aqueous dopamine solution was continuously injected into poly(dimethylsiloxane) microchannels. We found that various cells (fibrosarcoma HT1080, mouse preosteoblast MC3T3-E1, and mouse fibroblast NIH-3T3) predominantly adhered to PDA-modified regions, maintaining their normal morphologies. The cells aligned in the direction of striped PDA patterns, and this tendency was not limited by the type of cell line. Because PDA modification does not require complex chemical reactions and is applicable to any type of material, it enables cell patterning in a simple and versatile manner as opposed to conventional methods based on the immobilization of adhesive proteins. The PDA-based method of cell patterning should be useful in many biomaterial research areas such as the fabrication of tissue engineering scaffolds, cell-based devices for drug screening, and the fundamental study of cell-material interactions.