A method for producing a cell culture substrate to which cells adhere in a finely processed pattern while retaining the pattern for a long period of time to culture the cells and a production apparatus used in the production method. The method includes: forming a patterning substrate by forming: on a base material, a light shielding portion, and a cell adhesive layer having adhesion to a cell and containing a cell adhesive material which is decomposed or denatured by an action of a photocatalyst upon energy irradiation so as to cover the light shielding portion; an energy irradiating process of irradiating energy to the patterning substrate from the base material side to form a pattern; and a cell adhesion process of making the cell adhere to the cell adhesion portion in a cell culture medium containing the cell and a culture medium.