Background <p>Aloe-emodin, a type of anthraquinone derivative that exhibits diverse pharmacological activities, has been found to inhibit the proliferation of human hepatocellular carcinoma (HCC) cells despite concerns about hepatotoxicity; however, the specific mechanism underlying its anticancer activity remains poorly understood.</p> Objective <p>This study aimed to investigate the proapoptotic effects of aloe-emodin and examined its mechanism of action in the human HCC cell line Hep3B.</p> Results <p>Aloe-emodin significantly induced apoptosis, which was associated with impaired mitochondrial function, but not necrosis. Aloe-emodin treatment also triggered autophagy by upregulating the expression of microtubule-associated protein light chain 3 (LC3)-II and p62, and translocating them from the cytoplasm to the mitochondria. Moreover, aloe-emodin significantly increased the expression of the mitophagy inducers PTEN-induced kinase 1 (PINK1) and parkin in the mitochondria, suggesting the induction of autophagic flux in the mitochondria. However, 3-methyladenine, an autophagy blocker, neutralized these changes induced by aloe-emodin and counteracted aloe-emodin-induced cytotoxicity, suggesting that aloe-emodin-induced autophagy has a proapoptotic effect. Furthermore, aloe-emodin activated AMP-activated protein kinase (AMPK) signaling; however, the AMPK inhibitor compound C (CC) reversed the aloe-emodin-induced expression of mitophagy inducers and autophagy regulators in the mitochondria. The autophagy- and cytotoxicity-inducing activities of aloe-emodin were also abolished in the presence of CC.</p> Conclusion <p>The present findings demonstrate that aloe-emodin exerts cytotoxic effects on Hep3B cells by inducing mitochondrial autophagy through the activation of the AMPK pathway, indicating that aloe-emodin could be a potential therapeutic agent for HCC.</p>