The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill-defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D-glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [(3)H]-thymidine incorporation and cell-cycle regulatory protein expression levels compared with 5 mM D-glucose or 25 mM L-glucose. In addition, high glucose increased transforming growth factor-beta1 (TGF-beta(1)) mRNA and protein expression levels. High glucose-induced cell-cycle regulatory protein expression levels and [(3)H]-thymidine incorporation, which were inhibited by TGF-beta(1) siRNA transfection and TGF-beta(1) neutralizing antibody treatment. High glucose-induced phosphorylation of protein kinase C (PKC), p44/42 mitogen-activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time-dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10(-6) M; bisindolylmaleimide I, 10(-6) M), LY 294002 (PI3 kinase inhibitor, 10(-6) M), Akt inhibitor (10(-5) M), PD 98059 (p44/42 MAPKs inhibitor, 10(-5) M), SB 203580 (p38 MAPK inhibitor, 10(-6) M), and rapamycin (mTOR inhibitor, 10(-8) M) blocked the high glucose-induced cellular proliferation and TGF-beta(1) protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF-beta(1) expression via Ca(2+)/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways.