Aim: To investigate the ability of ox-LDL to induce ossification of endothelial progenitor cells (EPCs) in vitro and explored whether oxidative stress, especially hypoxia inducible factor-1 alpha (HIF-1 alpha) and reactive oxygen species (ROS), participate in the ossific process. Methods: Rat bone marrow-derived endothelial progenitor cells (BMEPCs) were cultured in endothelial growth medium supplemented with VEGF (40 ng/mL) and bFGF (10 ng/mL). The cells were treated with oxidized low-density lipoprotein (ox-LDL, 5 mu g/mL) and/or beta-glycerophosphate (beta-GP, 10 mmol/L). Calcium content and Von Kossa staining were used as the measures of calcium deposition. Ossific gene expression was determined using RT-PCR. The expression of osteocalcin (OCN) was detected with immunofluorescence. Alkaline phosphatase (ALP) activity was analyzed using colorimetric assay. Intercellular reactive oxygen species (ROS) were measured with flow cytometry. Results: BMEPCs exhibited a spindle-like shape. The percentage of cells that expressed the cell markers of EPCs CD34, CD133, and kinase insert domain-containing receptor (KDR) were 46.2%+/- 5.8%, 23.5%+/- 4.0%, and 74.3%+/- 8.8%, respectively. Among the total cells, 78.3%+/- 4.2% were stained with endothelial-specific fluorescence. Treatment of BMEPCs with ox-LDL significantly promoted calcium deposition, which was further significantly enhanced by co-treatment with beta-GP. The same treatments significantly increased the gene expression of core-binding factor a-1 (cbfa-1) and OCN, while decreased the gene expression of osteoprotegerin (OPG). The treatments also significantly enhanced the activity of ALP, but did not affect the number of OCN(+) cells. Furthermore, the treatments significantly increased ROS and activated the hypoxia inducible factor-1 alpha (HIF-1 alpha). In all these effects, ox-LDL acted synergistically with beta-GP. Conclusion: Ox-LDL and beta-GP synergistically induce ossification of BMEPCs, in which an oxidizing mechanism is involved.