Esophageal cancer is a highly lethal cancer with a rapidly increasing incidence and a poor prognosis. Atractylenolide I is a natural sesquiterpene lactone extracted from the rhizome of the Asteraceae plant, which has a variety of pharmacological effects, such as anti-inflammatory and immunomodulatory. Still, its impact on esophageal cancer has not been reported. Therefore, this study investigated the in vitro and in vivo effects of Atractylenolide I on the growth and proliferation of esophageal cancer and explored its possible mechanisms.To evaluate the effect of atractylenolide I on esophageal cancer cells, apoptosis assay and cell cycle assay tests were performed. Atractylenolide I was used to treat esophageal cancer cells for 48 hours, and flow cytometry detects apoptosis and cell cycle. The Wnt/β-catenin-related pathway proteins were then detected by Western blotting. For in vivo studies, an esophageal cancer graft tumor model was established subcutaneously in BALB/c nude mice, which were given Atractylenolide I treatment for 2 weeks.The result shows that Atractylenolide I inhibited the proliferation and induced apoptosis of esophageal squamous carcinoma and adenocarcinoma cells. Further research shows that Atractylenolide I inhibited the Wnt/β-catenin signaling pathway, decreased the expression of CCND1, MYC, and FN1 genes, and thus increased the apoptosis of esophageal cancer cells and blocked the cell cycle in G0/G1 phase, hence exerting the role of inhibiting esophageal cancer cells in vivo and in vitro.This study indicates that Atractylenolide I is an efficient lead compound for the treatment of esophageal cancer, providing a theoretical basis for further clinical development and application of Atractylenolide I.Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.