A method for manufacturing a cell culture substrate obtained by forming a coating layer of a polymer on the surface of a substrate, wherein the polymer is formed by polymerizing a monomer component containing a nitrogen atom-containing monomer represented by formula (I): wherein R1 is hydrogen atom or methyl group, R2 is an alkylene group having 1 to 6 carbon atoms, each of R3 and R4 is independently an alkyl group having 1 to 4 carbon atoms, R5 is an alkylene group having 1 to 4 carbon atoms, and Y is oxygen atom or —NH— group, includes irradiating the polymer with ion beam at a predetermined position of the coating layer of the polymer, to remove the coating layer of the polymer irradiated with the ion beam.