The involvement of caveolin-1 (Cav-1) and integrin β1 (IN β1) in regulation of embryonic stem (ES) cell growth by high glucose is by no means clear cut. Therefore, the aim of this study was to examine the influence of high glucose on Cav-1 and IN β1 expression in mouse ES cells and their signaling pathways to modulate proliferation. High glucose significantly increased Cav-1 and IN β1 expression. In addition, increased IN β1 expression was inhibited by Cav-1 small interfering RNA (siRNA). High glucose caused reactive oxygen species generation and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Inhibition of p38 MAPK blocked high glucose-induced Cav-1 and fibronectin (FN) expression. Moreover, phosphorylation of both Src and focal adhesion kinase (FAK) were increased by high glucose, which were inhibited by IN β1 antibody. In addition, high glucose increased the expression levels of PINCH1/2, integrin-linked kinase (ILK), and α-parvin [PIP] complex proteins, which were all inhibited by the FAK siRNA and Src specific inhibitor (PP2, 10−7 M). High glucose also increased F-actin expression, which was inhibited by ILK, PINCH1/2, and α-parvin siRNAs. Finally, high glucose-induced increase of ES cell proliferation was inhibited by TRIO and F-actin binding protein (TRIOBP) siRNA. The results demonstrate that high glucose-induced Cav-1 and IN β1 activation can stimulate ES cell proliferation through the modification of focal adhesion signaling pathways. J. Cell. Physiol. 226: 1850–1859, 2011. © 2010 Wiley-Liss, Inc.