Stellaria media (Linn.) Villars is a traditional Chinese medicine that has been used for over 200 years, mainly for the treatment of dermatitis and other skin diseases. It has also been used as an anti-viral agent. All the fresh chickweed juice samples used in this study were prepared using macroporous resin and ultrafiltration technology. The anti-hepatitis B virus (HBV) activity of S. media was evaluated in vitro using the human HBV-transfected liver cell line HepG2.2.15. The concentrations of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG2.2.15 cell culture medium were determined by enzyme-linked immunosorbent assay (ELISA) after S. media-n (SM-n) treatment for 6 or 9 days. HBV DNA was quantified using transcription-mediated amplification and real-time polymerase chain reaction. In HepG2.2.15 cells, 30 μg/mL SM-3 effectively suppressed the secretion of HBsAg and HBeAg with inhibition rates of 27.92% and 25.35% after 6 days of treatment, respectively. Consistent with the reduction in HBV antigens, SM-3 also reduced the level of HBV DNA in a dose-dependent manner. The characterization and quantitation of the chemical composition of SM-3 showed the presence of flavonoid C-glycosides, polysaccharides, and protein, which exhibited diverse antiviral activities. In conclusion, our results demonstrate that SM-3 possesses potential anti-HBV activity in vitro. This is the first report demonstrating the anti-HBV effects of S. media, which is currently under early development as a potential anti-HBV drug candidate.