BACKGROUND: Chronic infection with the hepatitis B virus (HBV) represents a significant global health concern. Baicalein, a naturally occurring flavone derived from the roots of Scutellaria baicalensis Georgi, has exhibited both anti-inflammatory and antiviral activities. S. baicalensis is extensively utilized in traditional Chinese medicine for the treatment of various liver disorders, including hepatitis. However, the specific anti-HBV effects of baicalein have not been fully elucidated. PURPOSE: This study aimed to investigate the inhibitory effects of baicalein on HBV and to elucidate its underlying mechanisms. MATERIALS AND METHODS: The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were measured using enzyme-linked immunosorbent assay (ELISA) kits. Quantification of HBV DNA was performed using quantitative real-time polymerase chain reaction (qRT-PCR). Western blot analysis was conducted to evaluate proteins involved in autophagy, lysosomal acidification, and autophagy-related signaling pathways. Immunofluorescence microscopy was utilized to assess autophagic flux and lysosomal acidification. RESULTS: Baicalein demonstrated significant inhibition of HBsAg, HBeAg, and HBV-DNA secretion in both in vivo and in vitro environments. Subsequent investigations revealed that baicalein disrupted the intracellular trafficking of the hepatitis B virus by inhibiting the CCDC88A-AKT-mTOR (Coiled coil domain containing protein 88A- protein kinase B-mammalian target of rapamycin) signaling pathway. Additionally, baicalein induced autophagy in HepG2 (Human hepatocellular carcinoma cell line 2) and HepG2.215 cell models. The anti-hepatitis B antigen effect of baicalein was partially attenuated when both early and late stages of autophagy were inhibited. A significant correlation was identified between the phosphorylation of AMPKα and the enhanced autophagy observed in baicalein-treated cells. CONCLUSIONS: This study elucidates a novel mechanism by which baicalein inhibits the hepatitis B virus (HBV). Specifically, baicalein exerts its antiviral effects by activating autophagy and suppressing the CCDC88A-AKT-mTOR signaling pathway.