Interferon-α (IFN-α) inhibits the replication of hepatitis B virus (HBV) in vivo and in vitro, but the molecular mechanism of this inhibition has been elusive. We found that while HBV replication in transfected human hepatoma Huh-7 cell was severely inhibited by IFN-α treatment as reported previously, this inhibition was markedly impaired in the cell in which the expression of IFN-inducible, double-stranded RNA-dependent protein kinase (PKR) was stably and specifically suppressed through RNA-interference. Intracellular level of viral capsids was down-regulated likewise in a PKR-dependent manner, whereas that of HBV transcripts including the viral RNA pregenome was not affected by IFN-α treatment. Ectopic expression of PKR also resulted in the reduction of viral capsids with concomitant increase of phosphorylated eIF2α. These results suggested that PKR functions as a key mediator of IFN-α in opposing HBV replication, most likely through the inhibition of protein synthesis.