Exploiting the RNA interference pathway has shown promise for developing novel and effective treatment of hepatitis B virus (HBV) infection. To advance this approach, we analyzed the antiviral efficacy of a panel of 10 Pol III U6 promoter-encoded short hairpin RNAs (shRNAs) that target conserved sequences of the oncogenic HBx open reading frame. To facilitate intracellular processing, the shRNAs included mismatches in the 25-bp stem region and a terminal loop of miRNA-23. Two shRNAs (shRNA 5 and shRNA 6) showed knockdown of HBV markers by 80-100% in transfected hepatocytes and also in a murine hydrodynamic injection model of HBV replication. Intracellular processing of hairpin RNA with the intended strand bias correlated with antiviral efficacy. Moreover, markers of HBV replication were inhibited without inducing genes associated with the nonspecific interferon response. To assess the antiviral efficacy of the shRNAs in a context that is similar to natural HBV infection, shRNA-encoding cassettes were tested against the virus in a HBV transgenic murine model. When delivered using recombinant adenovirus vectors, U6 shRNA 5 and U6 shRNA 6 mediated significant HBV knockdown. Collectively, these observations indicate that U6 shRNA 5 and U6 shRNA 6 are promising candidates for therapy of chronic HBV infection.