Small interfering RNA (siRNA) has been shown to be active to inhibit the hepatitis B virus gene expression and replication in transient and stable transfection systems. Here in primary hepatocytes prepared from naturally woodchuck hepatitis virus (WHV)-infected woodchucks, four siRNAs targeting the WHV preS1, S, C, and X region led to a depletion of WHV transcripts and replicative intermediates with different kinetics and a decreased production of viral particles. Two siRNAs targeting WHV S and X region had the highest efficacy to deplete 70% of WHV transcripts and replicative intermediates. In addition, siRNA-mediated suppression of WHV enhanced the expression of cellular genes like MxA and MHC I. Specific siRNAs are able to inhibit the hepadnaviral replication and enhance the expression of cellular genes relevant for antiviral actions. Thus, siRNAs might be useful as novel antiviral agents for the treatment of chronic HBV infection.