Development of cell-patterning techniques is a major challenge for the construction of functional tissues and organs in tissue engineering. Recent progress in surface chemistry has enabled spatial control of cell adhesion onto cultural substrates by varying hydrophilicity, for example, by using poly (ethylene glycol) (PEG). In the present study, we developed a novel cell-patterning procedure using PEG-modified magnetite particles (PEG-Mags) and magnetic force. Using an array-patterned magnet, PEG-Mags were magnetically patterned on the surface of a tissue culture dish. The resultant substrate surface consisted of two regions: the PEG-Mag surface that acts as a cell-resistant region and the native substrate surface that promotes cell adhesion. When human keratinocyte HaCaT cells were seeded onto the PEG-Mag-patterned surface, cells adhered only to the native substrate surface, resulting in cell-patterning on the tissue culture dish. The patterned PEG-Mags were then washed away to expose the native substrate surface, and thereafter, when mouse myoblast C2C12 cells were seeded to the dish, cells adhered to the exposed substrate surface, resulting in a patterned coculture of heterotypic cells. Moreover, it is worth noting that the magnetic force-based cell-patterning procedure is not limited by the property of cultural substrate surfaces, and that cell-patterning of mouse fibroblast NIH3T3 cells on a monolayer of HaCaT cells was successfully achieved using PEG-Mags and magnetic force. These results indicate that this procedure provides a novel concept for cell-patterning and may be useful for tissue engineering and cell biology.