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Hepatocellular carcinoma (HCC), a common liver malignancy worldwide, has high morbidity and mortality. β-Thujaplicin, a tropolone derivative, has been used in some health-care products and clinical adjuvant drugs, but its use for HCC is unknown. In this study, we found that β-Thujaplicin inhibits the growth of HCC cells, but not normal liver cells, with nanomolar potency. Mechanistically, we found that β-Thujaplicin could induce autophagy, as judged by western blot, confocal microscopy, and transmission electron microscopy. Further using β-Thujaplicin combined with an autophagy blocker or agonist treatment HepG2 cells, we found that β-Thujaplicin induced autophagic cell death (ACD) mediated by ROS caused inhibition of the Akt-mTOR signaling pathway. Moreover, β-Thujaplicin triggered HepG2 apoptosis and increased cleaved PARP1, cleaved caspase-3, and Bax/Bcl-2 ratio, which indicated that β …