We propose a novel concept for cellular scaffolds with 2D-patterned mechanical properties. Thin films of glassy polystyrene (PS) with thicknesses ranging from 100 nm to 1 μm were prepared on epoxy resin-based line and space (L&S) patterned substrates. Although the outermost surface of PS on the L- and S-regions was sufficiently flat at the same level, the mechanical responses differed depending on the presence of the underlying resin foundation. The initial cell adhesion and spreading and the proliferation on the scaffolds were affected by the 2D-patterned mechanical properties, that is, cellular behavior was suppressed on mechanically unstable S-regions.