An understanding of cell–substrate interactions can provide insight into the complex interactions between the cell and its natural environment. Geometrical surface properties can influence the cell adhesion behavior, which is a fundamental process directly involved in cell growth, cell migration and cell differentiation. In this study, the phenomenon of cells displaying preference toward surface with irregular roughness generated by polystyrene islands (0.2m in diameter) has been investigated. Fibroblasts on surfaces of different roughness exhibited clear preference toward the rougher region, with larger cell spreading area and the degree of stretching. This preference was maintained even when the extracellular matrix (ECM) proteins were eliminated from the surface. Cells on the surfaces with irregular ECM protein coating did not display any preference, and showed insignificant differences in morphology. Our results reveal that geometrical surface properties can regulate cell adhesion behavior, which can lead to extensive understanding of how cells recognize surface features and react to them.