Estradiol-17 (E₂) is a steroid hormone, well-known for its roles in the regulation of various cell functions. However, the precise role E₂ plays in the proliferation of human mesenchymal stem cells (hMSC) has not been completely elucidated. In the present study, we examined the effects of E₂ on cell proliferation and the related signaling pathways using hMSCs. We showed that E₂ , at 10^-9 M, significantly increased [³H]thymidine incorporation after 24 hr incubation, and E₂ also increased [³H]thymidine incorporation at > 6 hr. Also, E₂ significantly increased the percentage of the cell population in the S phase based on FACS analysis. Moreover, E₂ increased estrogen receptors (ERs), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules were involved in an E₂-induced increase of [³H]thymidine incorporation. E₂ also increased hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) protein levels. These levels of protein expression were inhibited by ICI 182,780 (10_-6 M, an ER antagonist), staurosporin and bisindolylmaleimide I (10_-6 M, a PKC inhibitor), LY 294002 (10_-6 M, a PI3K inhibitor), Akt inhibitor (10_-5 M), SP 600125 (10_-6 M, a SAPK/JNK inhibitor), and PD 98059 (10_-5 M, a p44/42 MAPKs inhibitor). In addition, HIF-1 siRNA and ICI 182,780 inhibited E₂-induced VEGF expression and cell proliferation. VEGF siRNA also significantly inhibited E₂-induced cell proliferation. In conclusion, E₂ partially stimulated hMSC proliferation via HIF-1 activation and VEGF expression through the PKC, PI3K/Akt, and MAPKs pathways.