Hepatitis B virus X (HBX) protein is required for the productive infection of hepatitis B virus (HBV) in vivo and implicated in the development of hepatocellular carcinoma. We have previously shown that hTid-1 and Hdj1, the human Hsp40/DnaJ chaperone proteins, bind the HBV core protein and inhibit viral replication in cell culture system. Here, we report evidences to suggest that HBX is the major target of Hdj1 in the inhibition of HBV replication. Expression of Hdj1 in cultured human hepatoma HepG2 cells facilitated degradation of HBX by the proteasome pathway, and thereby inhibited replication of the wild-type HBV as well as that of the HBX-deficient mutant virus rescued by HBX supplied in trans. Mutational analyses indicated that J domain of Hdj1 is required for the process. These results might provide a molecular basis for the antiviral effect of cellular chaperones.