We developed a new facile method for the preparation of temperature-responsive cell culture surfaces (TRCS) by photo-polymerization of N-isopropylacrylamide using a surface-immobilized photoinitiator. In the first step, thioxanthone groups, which served as the photoinitiator, were directly formed on polystyrene culture dish surfaces (TX-PSt). Second, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto the TX-PSt surfaces (PIPAAm-TX-PSt) by using the visible light or light-emitting diode irradiation-induced surfaceinitiated polymerization method. By optimizing the amount of grafted PIPAAm, the resulting PIPAAm-TX-PSt successfully exhibited characteristics of a TRCS. In addition, a striped micropatterned polyacrylamide area was successively formed on the photoirradiated PIPAAm-TX-PSt surface by using a chromium photomask. These results show that temperature- responsive cell culture surfaces can be prepared through this new method.