Lipid rafts are cholesterol-rich microdomains of cell membranes that have a variety of roles in cellular processes including receptor-mediated signal transduction. Lipid rafts also occur in embryonic stem (ES) cells, but their role in ES cells is largely unknown. Therefore, we investigated the role of lipid rafts in the maintenance of ES cell self-renewal. In the present study, we observed that the presence of lipid rafts/caveolae. The results from sucrose gradient fractionation showed that the expression of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor beta (LIFRbeta) was decreased by treatment with methyl-beta-cyclodextrin (Mbeta-CD) but, interestingly, was not affected by caveolin-1 small interfering RNA (siRNA). In addition, LIF increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) and Akt, and the expression level of c-Myc, which were attenuated by the pretreatment with Mbeta-CD. However, caveolin-1 siRNA did not influence LIF-induced phosphorylation of STAT3 and Akt, and expression of c-Myc. Treatment with Mbeta-CD and caveolin-1 siRNA decreased expression levels of Oct4 protein and Oct4, Sox2, FoxD3, and Rex1 mRNAs in normal culture conditions. Additionally, Mbeta-CD and caveolin-1 siRNA decreased the expression levels of cyclin D1 and cyclin E, and the proliferation index [(S + G2/M)/(G0/G1 + S + G2/M)] of ES cells. We conclude that lipid raft/caveolae structures play important roles in the self-renewal of ES cells.