Background: Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is derived from herbal medicines and proved to have a strong antimicrobial activity. However, its anti-virus effects are less known. The aim of the present study was to investigate the effects of emodin, interferon alpha (IFN alpha), and lamivudine (3TC) on hepatitis B virus (HBV) in vitro. Material/Methods: The human hepatoma G2.2.15 cell line stably expresses hepatitis B virus particles in culture. The cells were exposed to different concentrations of emodin, IFN alpha, and lamivudine triphosphate, respectively. MTT (methyl thiazolyl tetrazolium) assay was used to evaluate the cytotoxicity of the drugs and real-time PCR was applied to quantify extracellular HBV DNA. HBsAg and HBeAg were assessed by enzyme-linked immunosorbent assay (ELISA). Results: The results showed that exposure of HepG2.2.15 cells to emodin resulted in a time- and concentration-dependent inhibition of HBV DNA replication and HBsAg secretion. After exposed to three different concentrations of emodin for 3, 6, and 9 days, the inhibition rates of extracellular HBV DNA, HBsAg, and HBeAg of each concentration decreased significantly (P < 0.05). After 9 days of treatment, the inhibition rates of extracellular HBV DNA of the different concentrations differed greatly (P < 0.001). IFN alpha and 3TC had similar inhibition results to HBV DNA replication to those previously found. Conclusions: These findings suggest that emodin may prove, to be a new modality to treat hepatitis B infection.