Recently drug-induced senescence has gained momentum as a new approach in cancer therapy. It is accepted that senescent cells display typical phenotypic features including flattened, enlarged, and multinucleated cell morphology. However, it is not well elucidated how these morphological alterations occur. The current study evaluates the possible role of Rho/Rho kinase pathway in cardiac glycoside-induced senescent cell morphology in HeLa cells. Our results indicate that the administration of cardiac glycosides, ouabain, digoxin, bufalin, to HeLa cells induced cellular senescence leading to an increase in the volume, area and maximum thickness of the cells. Although preincubation of specific Rho kinase inhibitor Y-27632 did not inhibit the occurrence of cardiac glycoside-induced senescence in cells, it reduced the cell area and cell volume. Inhibition of Rho by CT04 produced similar results as seen for the preincubation of Y-27632. In addition, inhibition of Rock caused a decrease in increased actin stress fibers in senescent cells induced by ouabain. Additionally, preincubation of Y-27632 decreased the ouabain-induced the phosphorylation of MYPT and cofilin. In conclusion, Rock inhibition-mediated alteration of senescent cell morphology may be associated with the decreased actin stress fibers formation. Since it is known that secretory activity is accompanied by the changes of cell morphology, these morphological alterations observed by the inhibition of Rho/Rho kinase pathway may also lead to important secretory functions of senescent cells.