Pharmacological modulation of cellular proteins as a means to block virus replication has been proposed as an alternative antiviral strategy that may be less susceptible than others to the development of viral drug resistance. Recent evidence indicates that the ubiquitin-proteasome pathway interacts with different aspects of the hepatitis B virus (HBV) life cycle in cell culture models of virus replication. We therefore examined the effect of proteasome inhibition on HBV replication in vivo using HBV transgenic mice. The proteasome inhibitor bortezomib (Velcade) inhibits proteasome activity in vivo and is used therapeutically for the clinical treatment of multiple myeloma. We found that a single intravenous dose of 1 mg of bortezomib/kg of body weight reduced virus replication for as long as 6 days. The inhibition of HBV by bortezomib was dose dependent and occurred at a step in replication subsequent to viral RNA and protein expression. The reduction in HBV replication did not result from nonspecific hepatocellular toxicity and was not mediated indirectly through the induction of an intrahepatic interferon response. Thus, pharmacological manipulation of the ubiquitin-proteasome pathway may represent an alternative therapeutic approach for the treatment of chronic HBV infection.