The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.