Cholesterol is a major lipid component of mammalian cells which regulates dynamical and physical properties of the plasma membrane. Lipid rafts are cholesterol- and sphingolipid-enriched regions of membrane that are implicated in various cellular responses. Considering the major impact of cholesterol in the mechanical properties of lipid bilayer, the role of lipid rafts and their integrity in regulation of cellular mechanotransduction is of peculiar interest. Cholesterol-depleting treatment of cells with methyl-beta-cyclodextrin (MbCD) significantly suppressed the activation of mechanosensitive (MS) stretch-activated ion channels in human myeloid leukemia K562 cells. Particularly, the open state probability (Po) of MS channels significantly decreased (Po=0.08±0.03 after MbCD treatment vs. 0.28±0.03 in control) whereas the minimal level of stimulus, that was needed for channel activation increased (70 80 mm Hg after MbCD vs. 30 40 mm Hg in control). Atomic force microscopy revealed the increase of plasma membrane stiffness after cholesterol depletion. The observed alteration of mechanical properties of the membrane and the inhibition of MS channels after MbCD treatment could not be explained by the change of dynamic properties of lipid bilayer after cholesterol sequestration. We hypothesized, that this effect was mediated by actin cytoskeleton rearrangement after lipid raft disruption. The disruption of rafts was confirmed by fluorescent staining of lipid raft marker GM1 ganglioside. Importantly, fluorescent data revealed the formation of F-actin network in K562 after cholesterol depletion. Importantly, the high level of MS channel activity in cholesterol-depleted cells was fully restored after F-actin disruption with cytochalasin D or latrunculin B. Experiments on different cell lines showed that the effect of cholesterol depletion on actin organization is determined by the initial state of microfilament system in the cell. Particularly, in cultured normal BALB/3T3 fibroblasts that characterized with highly developed microfilament system, the cholesterol depletion resulted in actin disassembly and reduction of stress fibers. On the contrary, in transformed 3T3B-SV40 fibroblasts, containing low amount of fibrillar actin, MbCD treatment induced intensive formation of stress fibers and increased cell spreading. Thus, the functional impact of cholesterol depletion and lipid raft disruption on actin cytoskeleton can be determined by an initial balance of fibrillar and globular actin. Cholesterol-regulated actin rearrangement may affect different signaling processes in living cells, including cellular mechanotranduction. Particularly, the assembly of F-actin network in K562 cells after cholesterol depletion increases the stiffness of plasma membrane and thus inhibits MS channel activity.