Background: Monoclonal antibodies to tumor endothelial cells (TECs) hold great promise for cancer angiogenesis-targeted therapy. The aim of the present study was to develop such an agent for esophageal cancer treatment. Materials and Methods: BALB/c mice were immunized with human esophageal tumor endothelial cells (ETECs) cultured with tumor homogenate. MAbs were produced, screened by immunofluorescence and immunohistochemistry (IHC) and an IgG1 kappa mAb 4B3 was selected. The mAb 4B3 antigen was analyzed by IHC and Western blotting. The antibody's effects on ETECs were determined by adhesion and tube formation assays, while its therapeutic potential was evaluated with a tumor model established by co-inoculating mice with the human esophageal cancer cell lines KYSE180 and ETECs. Results: MAb 4B3 recognized a 40-kDa surface antigen preferentially expressed on TECs and other stromal cells in human malignant tissues of esophagus, stomach, colon, liver, lung and breast compared with their normal counterparts. The antigen was not detected on cancer cells or normal epithelia in these tissues, nor was it detectable on any cells in the mouse xenografts of KYSE 180, including the host endothelia. MAb 4B3 inhibited ETEC adhesion to extracellular matrix proteins and tube formation in vitro. The antibody inhibited angiogenesis and growth of the tumor formed by co-inoculation. Conclusion: These results suggest that mAb 4B3 has therapeutic potential for esophageal cancer.