A main object of the invention is to provide a new method for producing a cell culture substrate used to cause cells to adhere in a highly precise form onto a base material and then culture the cells.To attain the object, the invention provides a method for producing a cell culture substrate comprising: a patterning substrate forming process of forming, on a base material, a cell culture patterning layer wherein a cell adhesion portion having cell adhesive properties and a cell adhesion-inhibiting portion having cell adhesion-inhibiting properties can be formed by action of a photocatalyst by energy irradiation, thereby forming a patterning substrate; an energy irradiating process of irradiating the energy onto the cell culture patterning layer, thereby forming the cell adhesion portion and the cell adhesion-inhibiting portion in a pattern form by action of the photocatalyst; and a cell-containing liquid applying process of applying a cell-containing liquid onto the cell adhesion portion by a region-selecting applying method of applying the cell-containing liquid selectively onto the patterned cell adhesion portion.