Ampelopsis sinica root is widely used in Chinese folk medicine for treating liver disorders caused by the hepatitis B virus (HBV). The present study was performed in order to investigate the anti-HBV activity and mechanisms of the ethanol extract from A. sinica root (EASR) in vitro. The antiviral activity of EASR was examined by detecting the levels of HBsAg, HBeAg and extracellular HBV DNAs in stable HBV-producing human hepatoblastoma HepG2 2.2.15 cells. We found that EASR effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, and it also suppressed the amount of extracellular HBV DNA. After EASR treatment, the percentage of apoptotic cells was found to be significantly higher than that of control by flow cytometric analysis. A luciferase reporter gene assay was used to determine the effects of EASR on the activities of HBV promoters and intracellular signaling pathways. The results showed that EASR selectively inhibited the activities of HBV promoters (Cp, S1p and Fp) and the p53 signaling pathway in HepG2 cells significantly. These data indicate that EASR exerts anti-HBV effects via inhibition of HBV promoters and the p53-associated signaling pathway, which helps to elucidate the mechanism underlying the potential therapeutic value of EASR.