Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Although a preventive vaccine is available, the therapeutic options for chronically infected patients are limited. It has been shown that RNA interference can prevent HBV gene expression and replication in vivo when HBV expression vectors are delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. However, the therapeutic potential of siRNAs to interrupt ongoing HBV replication in vivo has not been established. Here, we show that expression of HBV-specific siRNAs in the liver of HBV transgenic mice by recombinant adenoviruses can suppress preexisting HBV gene expression and replication to almost undetectable levels for at least 26 days. These results demonstrate that efficiently delivered siRNAs should be able to silence HBV in chronically infected patients.