In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we developed a novel technique to control living adherent cells on a culture substrate by irradiating light onto multiple points simultaneously. In clear contrast to the conventional cell patterning using the previously patterned substrate, the cell-retaining area can be defined even after cell seeding, and the captured cells can continue to grow freely beyond the defined area afterwards. Also the patterned co-culture of living cell was demonstrated by introducing living cells sequentially onto patterned cell adhesion areas assigned and created in each step on a newly developed photo-responsive culture substrate.