A noncytotoxic procedure for the spatial organization of multiple cell types remains as a major challenge in tissue engineering. In this study, a simple and biocompatible micropatterning method of multiple cell types on a polymer surface is developed by using ion implantation. The cell-resistant Pluronic surface can be converted into a cell-adhesive one by ion implantation. In addition, cells show different behaviors on the ion-implanted Pluronic surface. Thus this process enables the micropatterning of two different cell types on a polymer substrate. The micropatterns of the Pluronic were formed on a polystyrene surface. Primary cells adhered to the spaces of the bare polystyrene regions separated by the implanted Pluronic patterns. Secondary cells then adhered onto the implanted Pluronic patterns, resulting in micropatterns of two different cells on the polystyrene surface.