The molecular events of hyperglycemia-triggered increase in adipogenic induction of lipid accumulation remain unclear. We examined the effects of hyperglycemia on adipogenic induction of lipid accumulation and its involved signaling molecules such as phosphoinositide 3-kinase (PI3K), extracellular signal-regulated protein kinases (ERK), and peroxisome proliferator-activated receptor (PPAR). Bone marrow-derived mesenchymal stem cells (MSCs) isolated from FVB/N mice were capable of differentiating into adipocytes in adipogenic medium. The effects of high glucose (HG, 25.5 mM) were assessed in vitro by RT-PCR, ELISA, flow cytometry, immunostaining and immunoblotting. The in vivo effect of hyperglycemia was further studied in streptozotocin-induced diabetic FVB/N mice. Exposure of MSCs to HG enhanced adipogenic induction of lipid accumulation as compared with 5.5 mM glucose. HG increased PPAR expression and PI3K activity and its downstream effector Akt phosphorylation during adipogenesis. Inhibition of PI3K/Akt activity with PI3K inhibitor LY294002 or by expressing the dominant negative p85 or Akt prevented the HG-enhanced PPAR-dependent adipogenic induction of lipid accumulation. Moreover, HG increased the phosphorylation of ERK1/2 during adipogenesis. Mitogen-activated protein kinase (MAPK)/ERK inhibitor PD98059 inhibited the PI3K activity, Akt phosphorylation and lipid accumulation triggered by HG. PI3K inhibitor LY294002 didn't affect the HG-increased ERK1/2 phosphorylation during adipogenesis. We next observed that adipogenic induction of lipid accumulation of MSCs isolated from streptozotocin-induced diabetic mice is enhanced. Moreover, triglyceride, PPAR expression, phosphorylated Akt and ERK1/2, and marrow fat in bones of streptozotocin-diabetic mice were also increased. These results suggest that hyperglycemia enhances the adipogenic induction of lipid accumulation through an ERK1/2-activated PI3K/Akt-regulated PPAR pathway.