Scanning probe microscopy and immunofluorescence observations indicated that cellular stiffness was attributed to a contractile network structure consisting of stress fibers. We measured temporal variations in cellular stiffness when cellular contractility was regulated by dosing with lysophosphatidic acid or Y-27632. This experiment revealed a clear relation between cellular stiffness and contractility: Increases in contractility caused cells to stiffen. On the other hand, decreases in contractility reduced cellular stiffness. In both cases, not only the stiffness of the stress fibers but also that of the whole of the cell varied. Immunofluorescence observations of myosin II and vinculin indicated that the stiffness variations induced by the regulation of cellular contractility were mainly due to rearrangements of the contractile actin network on the dorsal surface. Taken together, our findings provide evidence that the actin cytoskeletal network and its contractility features provide and modulate the mechanical stability of adherent cells.