Controlling the spatial organization of cells is a critical step toward engineering tissues with distributed networks of blood vessels or nerve cells. Here we report a new soft-lithography-based approach for micropatterning proteins and cells on the surface of biodegradable chitosan substrates that are more applicable to engineering tissues than the gold, silver, glass, or silicone substrates currently used in cell micropatterning studies. In this approach, we use random copolymers of oligo(ethylene glycol) methacrylate (OEGMA), which resists protein and cell adsorption, and methacrylic acid (MA), which adheres strongly onto the chitosan substrate via acid−base interactions, to form stable protein and cell resistant micropatterns on chitosan surfaces. At optimal ratios of OEGMA to MA, copolymers are formed that exhibit superior long-term resistance to protein adsorption and cell adhesion even under cell culture conditions. Spatial control of cell organization and alignment using OEGMA/MA micropatterned chitosan is demonstrated using human microvascular endothelial cells.