AbstractAdvanced gastric cancer (GCa) remains highly lethal due to the lack of effective therapies. Identifying promising therapeutic targets and developing effective treatment against GCa are urgently needed. Through mRNA and protein analysis of GCa clinical tumor samples, we found that autophagy-related gene 4B (ATG4B) was overexpressed in GCa tumors and that its high expression was associated with patients’ poor prognosis. Knockdown of ATG4B significantly inhibited GCa cell survival and tumor growth. To further probe the role of ATG4B in GCa by pharmacological means, we screened an in-house marine natural compound library against ATG4B and identified Azalomycin F4a (Am-F4a) as a novel and potent ATG4B inhibitor. Am-F4a directly bound to ATG4B with high affinity and effectively suppressed GCa cell autophagy via inhibition of ATG4B both in vitro and in vivo. Moreover, Am-F4a or ATG4B knockdown significantly suppressed tumor growth as well as GCa cell migration and invasion. Am-F4a effectively blocked the metastatic progression of primary GCa and sensitized tumors to chemotherapy. Taken together, our findings indicate that ATG4B is a potential therapeutic target against GCa and the natural product Am-F4a is a novel ATG4B inhibitor that can be further developed for the treatment of GCa.