Membrane-cytoskeleton interaction regulates trans-membrane currents through stretch activated channels (SACs); however the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence and patch clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables which, tensed by myosin II motor, activate SACs by modifying the topography, the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, C_m) properties of the cell surface. Stimulation with sphingosine 1-posphate (S1P) to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeletal-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, membrane capacitance, C_m used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce C_m and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.