<b>Abstract</b>— <p>Piezo1 channels are mechanically activated (MA) cation channels that play an important role in skeletal muscle physiology. However, the specific mechanisms of Piezo1 regulation in skeletal muscle are not yet fully understood. At the same time, the activity of different ion channels is known to be dependent on lipid rafts, which are dynamic microdomains in the cell membrane enriched in sphingolipids and cholesterol. Therefore, the aim of this study was to evaluate the role of lipid rafts in the muscle-specific regulation of Piezo1 using a cholesterol-removing agent, methyl-beta-cyclodextrin (MβCD), and the sphingolipid sphingosine-1-phosphate (S1P) in a C2C12 myoblast model. Fluorescence labeling and intracellular calcium measurements demonstrated that the disruption of lipid rafts with MβCD resulted in disassembly of the initially present actin cytoskeleton in C2C12 cells and decreased Piezo1-mediated Ca²⁺ influx into the cells. In contrast, stimulation of myoblasts with S1P resulted in increased formation of lipid rafts and actin stress fibers, as well as enhanced Ca²⁺ influx through Piezo1 channels. These findings suggest the involvement of lipid rafts in the regulation of Piezo1 activity in C2C12 myoblasts, which may be mediated by lipid raft components themselves and/or by lipid raft-induced rearrangements of the actin cytoskeleton.</p>