<p>Skeletal muscle function is critically dependent on the metabolism of cholesterol and its sarcolemmal levels. Decreased cholesterol availability is associated with various pathological conditions, causing muscle weakness. Here, we tested the hypothesis that a reduction in cholesterol content can affect the resting membrane potential, an essential parameter for membrane transport and electro-mechanical coupling in fibers of mouse diaphragm, the main respiratory muscle. 20-min exposure to methyl-β-cyclodextrin (MβCD) concentration-dependently reduced the level of muscle cholesterol. This effect was more pronounced in surface muscle fibers, where MβCD at a concentration of 30 mM decreased cholesterol content by 40% and depolarized the membranes by approximately 20 mV in both junctional and extrajunctional regions of the muscle fibers. MβCD-induced depolarization was inhibited by approximately 20%, 19%, 34%, and 42% with GIIIB µ-conotoxin (Nav1.4 channel inhibitor), nitrendipine (L-type Ca²⁺ channel blocker), A-784,168 (TRPV1 antagonist), and ruthenium red (nonselective blocker of Ca²⁺ channels), respectively. MβCD increased sarcoplasmic Ca²⁺ levels at resting conditions and interfered with sarcolemma integrity in some muscle fibers. Finally, 30 mM MβCD aggravated muscle fatigue and disrupted the recovery of the contraction force at direct stimulation. These results indicate that plasma membrane cholesterol is essential for the maintenance of resting membrane potential due to restricting the activity of Ca²⁺ (TRPV1, Cav1.1) and Na⁺ (Nav1.4) channels as well as keeping membrane integrity in skeletal muscle. Cholesterol depletion-induced membrane depolarization might be a potential reason for muscle weakness.</p>