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There is a great demand for effective cell separation techniques that do not require the labeling of cell surfaces for applications in cell transplantation therapy and cell analysis. In the present study, we prepared thermoresponsive convex or concave substrates with circular hole, cylindrical pillar, and line patterns of various sizes as thermally modulated cell separation materials through the combination of thermal nano-imprinted lithography and subsequent surface-initiated atom transfer radical polymerization of poly(N-isopropylacrylamide). Three types of human cells, human umbilical vein endothelial cells, normal human dermal fibroblasts, and human skeletal muscle myoblast cells, which are commonly used in cardiovascular tissue engineering, were employed in this study. All three cell types could adhere to the prepared thermoresponsive micro- or nano-imprinted substrates at 37 °C and detached at 20 °C. The specific cell adhesion and detachment properties were different for each cell type, and they could be altered simply by changing the pattern shapes and sizes of the surface. In particular, large differences between the three cell types were obtained on the 2 μm hole pattern. Using this difference in cell adhesion properties, thermally modulated cell separation application was achieved by successively incubating at 37 °C and 20 °C. Thus, our thermoresponsive micro/nano-imprinted substrates can be utilized as cooperative cell separating materials by combining appropriate convex or concave patterns and mild temperature changes.