Background/Aims: β-Dystroglycan (β-DG) is a transmembrane glycoprotein that links the intracellular cytoskeleton to the extracellular matrix and is crucial for the molecular pathway of lateral force transmission in muscle. We aimed to investigate the effect of decreasing sarcolemmal cholesterol on the distribution of β-DG, its interaction with dystrophin and the impact on the contraction efficiency of muscle. Methods: Isolated rat extensor digitorum longus muscles were incubated with methyl β-cyclodextrin (MβCD) to deplete cholesterol and with MβCD-cholesterol to restore cholesterol. Electric stimulation protocols were used to determine muscle force and fatigue. Detergent-resistant membranes (lipid rafts) were separated from isolated skeletal muscle sarcolemma. The distribution and interactions of β-DG, caveolin-3 and dystrophin were determined by an immunoreactivity analysis. Results: Cholesterol depletion in muscle results in a weakened force of contraction, which recovers after cholesterol restoration. The rate of fatigue is unaffected, but fatigue recovery is dependent upon cholesterol restoration. MβCD modifies the structures of lipid rafts obtained from MβCD-treated muscles by, displacing the membrane proteins β-DG and caveolin-3 f from the lipid raft, thus reducing the interaction of β-DG with dystrophin. Conclusion: Cholesterol depletion weakens the muscle contractile force by disturbing the sarcolemmal distribution of β-dystroglycan and its interaction with dystrophin, two key proteins in the alignment of lateral force transmission pathway.