To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting HBV C gene region on hepatitis B virus (HBV) in vivo.An animal model of HBV infection was developed hydrodynamically, and pcDNA3.1-HBV and siRNA were together injected into the tail vein of the BALB/c mice. HBsAg was analyzed by time-resolved immunofluorometric assay, HBV DNA was analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR, and viral specific proteins (HBsAg and HBcAg) in the mice livers were assayed using immunohistochemical staining.In the mice, the siRNA effectively inhibited HBV replication and expression compared with the controls. The inhibitive effect of siRNA on HBV lasted at least 3 days.These results demonstrate that RNAi can substantially inhibit HBV replication and expression in vivo.