Osteosarcoma is a highly vascular and extremely destructive malignancy, and the survival of patients with osteosarcoma has not improved significantly in recent years. Antiangiogenic therapy currently holds great potential in conjunction with conventional treatment modalities for osteosarcoma. However, there are examples of gradual loss of response, and perhaps acquired resistance to antiangiogenic drugs. The acquired resistance of antiangiogenesis may be associated with a lot of hypoxia-response genes. The human apurinic/apyrimidinic endonuclease (Ape1) protein, a bifunctional redox factor and apurinic/apyrimidinic (AP) endonuclease, plays a crucial role in protecting against cell death due to hypoxia. We therefore hypothesized that Ape1 may contribute to the resistance of antiangiogenic therapy. To investigate the effect of Ape1 on the sensitivity of human osteosarcoma cells to endostatin, we constructed an Ape1 small interfering RNA expression vector, pSilenceApe1. Transfection of human osteosarcoma 9901 and HOS cells with pSilenceApe1 resulted in a dose-dependent loss of Ape1 protein. pSilenceApe1 also significantly suppressed the expression of vascular endothelial growth factor (VEGF) protein in the 9901 cells. Combined treatment with pSilenceApe1 and recombinant human endostatin (rhES) showed potent antiangiogenic effects in the transwell chamber invasion assay. Then, 20 nude mice bearing 9901 xenografts were divided into four groups: the phosphate-buffered saline treatment control group; the rhES treatment group (1.5 mg/kg, daily); the pSilenceApe1 treatment group (20 microg, once every 3 days); and the combination of rhES and pSilenceApe1 treatment group. pSilenceApe1 significantly suppressed the expression of Ape1 and VEGF protein in the 9901 xenografts. The tumor-inhibition rate of the pSilenceApe1, rhES, and combination of rhES and pSilenceApe1 treatment groups was 38.23, 35.29, and 62.18%, respectively. Furthermore, a significant decrease in microvessel density with an increase in apoptosis was observed following combined treatment with pSilenceApe1 and rhES, compared with control and either agent alone in 9901 xenografts. These results indicate that Ape1 small interfering RNA could enhance the sensitivity of osteosarcoma cells to endostatin.