The aim of this study was to investigate the effect of recombinant human endostatin (rh-Endo) in combination with radiation therapy (RT) on esophageal squamous cell carcinoma (ESCC) and explore the potential mechanisms. ECA109-bearing nude mice were administered RT and/or rh-Endo treatment. Tumor volume, survival, hypoxia and vascular parameters were recorded during the treatment schedule and follow-up as measures of treatment response. ESCC cell lines (ECA109 and TE13) and human umbilical vein endothelial cells (HUVECs) were developed to investigate the outcomes and toxicities of rh-Endo and RT in vitro. Hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were also evaluated. In vivo studies of ECA109-bearing xenografts showed that rh-Endo improved the radioresponse, with normalization of tumor vasculature and a reduction in hypoxia. In vitro studies showed that rh-Endo did not radiosensitize ESCC cell lines but did affect endothelial cells with a time- and dose-dependent manner. Studies of the molecular mechanism indicated that the improved radioresponse might be due to crosstalk between cancer cells and endothelial cells involving HIF and VEGF expression. Our data suggest that rh-Endo may be a potential anti-angiogenic agent in ESCC especially when combined with RT. The improved radioresponse arises from normalization of tumor vasculature and a reduction in hypoxia.