A substrate having a pattern of two or more materials exhibiting different grafting efficiencies for a temperature-responsive polymer that varies its interaction with water in a temperature range of 0 to 80° C. by electron beam irradiation under the same conditions is grafted with the temperature-responsive polymer by simultaneously irradiating the surfaces of the materials with electron beams to obtain a temperature-responsive cell culture substrate. According to this method, a temperature-responsive cell culture substrate having a surface (1) that allows cells to adhere thereto and to grow thereon during cell culturing and that allows the adhering and grown cells to be detached therefrom by changing the culturing temperature and a surface (2) that does not allow the cells to adhere thereto at all can be obtained by a simple process.