Aurora B kinase has emerged as a key regulator of mitosis and deregulation of Aurora B activity is closely related to the development and progression of human cancers. In the present study, we found that Aurora B is overexpressed in human esophageal squamous cell carcinoma (ESCC), high levels of Aurora B protein were associated with a worse overall survival rate in ESCC patients. Depleting of Aurora B blunted the malignant phenotypes in ESCC cells. Importantly, we demonstrated that a natural compound, deguelin, has a profound anti-tumor effect on ESCC via inhibiting Aurora B activity. Deguelin potently inhibited in vitro Aurora B kinase activity. The in silico docking study further indicated that deguelin was docked into the ATP-binding pocket of Aurora B. Inhibition of Aurora B activity attenuated growth of ESCC cells, resulted in G2/M cell cycle arrest, polyploidy cells formation, and apoptosis induction. Knocking down of Aurora B decreased the sensitivity of ESCC cells to deguelin. The in vivo results showed that deguelin blocked the phosphorylation of histone H3 and inhibited the growth of ESCC tumor xenografts. Overall, we identified deguelin as an effective Aurora B inhibitor, which deserves further studies in other animal models and ESCC treatment.