Bone is continuously remodeled by bone resorption and formation and, accordingly, bone metabolism is tightly regulated to maintain homeostasis. Deviation from the normal conditions of bone resorption can result in bone diseases. Lipid rafts are specialized plasma membrane microdomains that are enriched with glycosphingolipids, sphingomyelin and cholesterol. Lipid rafts are important for the transport of select membranes and relay stations involved in intracellular signaling.To investigate the role of lipid rafts in RAW264 cells signaling, lipid rafts were disrupted by depleting cholesterol through the introduction of methyl-β-cyclodextrin (MβCD). We found that sRANKL-induced differentiation into osteoclasts was markedly inhibited by MβCD in a dose-dependent manner. MβCD enhanced the sRANKL-induced phosphorylation of ERK1/2 MAP kinase. In contrast, MβCD blocked the phosphorylation of IκB. These findings suggest that cholesterol might play a crucial role in the regulation of the sRANKL-mediated signaling pathway and in osteoclast differentiation of RAW264 cells, thereby reflecting its importance in the formation of plasma membrane lipid rafts.