Diabetic patients tend to exhibit delayed bone formation and osteoblast differentiation, which results in osteopenia. Recently, numerous clinical reports suggest that 635-nm light irradiation improves bone regeneration and wound healing, and reduces pain in patients suffering from diabetes. The purpose of the present study was to test the hypothesis that 635-nm irradiation can influence bone formation by MC3T3-E1 osteoblasts cultured on high concentrations of glucose(25mmol/L D-glucose) in the presence or absence of phorbol 12-myristate 13-acetate(PMA), and to establish an in vitro pathological model of bone formation. The effect of 635-nm irradiation on bone formation was investigated using Alizarin Red S staining, and alkaline phosphatase enzyme activ ity and calcium deposition assays. In addition, gene expression of the o steogenic markers BMP-2, osterix and osteocalcin were assayed by RT-PCR. Calcium deposition by MC3T3-E1 cells was reduced in the presence of high concentrations of glucose or by PMA supplementation. However, 635-nm irradiation led to an increase in calcium deposition by MC3T3 cells, followed by increased bone mineralization. mRNA expression of BMP-2 and osterix at an early stage and of osteocalcin at a late stage was significantly upregulated by 635-nm irradiation in MC3T3-E1 cells supplemented with high concentrations of glucose. Irradiation at 635 nm increases bone mineralization in MC3T3-E1 cells cultured in vitro on high concentrations of glucose and alters osteogenic gene expression, which accelerates bone formation in hyperglycemic conditions.