Abstract Esophageal cancer incidence is increasing and has few treatment options. In studying receptor tyrosine kinases associated with esophageal cancers, we have identified EPHB4 to be robustly overexpressed in cell lines and primary tumor tissues. In total, 94 squamous cell carcinoma, 82 adenocarcinoma, 25 dysplasia, 13 Barrett esophagus, and 25 adjacent or unrelated normal esophageal tissues were evaluated by immunohistochemistry. EPHB4 expression was significantly higher in all the different histologic categories than in adjacent normal tissues. In 13 esophageal cancer cell lines, 3 of the 9 SCC cell lines and 2 of the 4 adenocarcinomas expressed very high levels of EPHB4. An increased gene copy number ranging from 4 to 20 copies was identified in a subset of the overexpressing patient samples and cell lines. We have developed a novel 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model of esophageal cancer that recapitulates the EPHB4 expression in humans. A specific small-molecule inhibitor of EPHB4 decreased cell viability in a time- and dose-dependent manner in 3 of the 4 cell lines tested. The small-molecule inhibitor and an EPHB4 siRNA also decreased cell migration (12%–40% closure in treated vs. 60%–80% in untreated), with decreased phosphorylation of various tyrosyl-containing proteins, EphB4, and its downstream target p125FAK. Finally, in a xenograft tumor model, an EPHB4 inhibitor abrogated tumor growth by approximately 60% compared with untreated control. EphB4 is robustly expressed and potentially serves as a novel biomarker for targeted therapy in esophageal cancers. Cancer Res; 73(1); 184–94. ©2012 AACR.