Esophageal cancer (EC) is among the severest cancers causing most fatalities around the world with an increasing incidence. Oleuropein exhibits anti-tumor properties in several human cancers. We aimed to investigate the effect of oleuropein in human EC, and to reveal the molecular target involved in EC tumorigenesis. Cell proliferation, migration and invasion assays were performed to assess the effect of oleuropein on EC cells. A xenograft tumor mouse model was utilized to assess the in vivo effect of oleuropein. Hypoxia-inducible factor-1α (HIF1α) and B-cell translocation gene 3 (BTG3) expressions were examined in oleuropein-treated EC cells. The regulatory effect of HIF1α on BTG3 mRNA was evaluated by chromatin immunoprecipitation and luciferase reporter assays. Oleuropein inhibited the growth of EC cells and xenograft EC tumor, as well as inhibiting HIF1α and upregulating BTG3 expressions. BTG3 mRNA expression was under hypoxia inhibition through the HRE in its promoter region. BTG3 knockdown abolished the inhibitory effect of oleuropein on EC cells in vitro, as well as on EC xenograft tumor in vivo. Oleuropein inhibits EC tumorigenesis through hypoxic suppression of BTG3 mRNA, supporting the clinical application of oleuropein, and HIF1α and BTG3 mRNA as potential molecular targets in treatment against EC.