Background Toll-like receptors (TLRs) are a key component of the innate immune system and TLR2 has been shown to be involved in the immunopathogenesis of hepatitis B virus (HBV) infection in vivo. We investigated the role of TLR2 stimulation of virus-infected hepatocyte cell lines as a potential antiviral mechanism in vitro. Methods The hepatoblastoma cell line HepG2 was transduced with recombinant HBV baculoviruses and the hepatoma cell line Huh-7 was transiently transfected with complimentary DNA clones of HBV. HBV viral replication was quantified after stimulation with interleukin (IL)-1β and Pam-2-Cys, a synthetic TLR2 ligand, by measuring intracellular core-associated single-stranded HBV DNA using Southern blot hybridization, as well as viral nucleocapsid formation using a non-denaturing immunoblot method. Results Stimulation of both cell lines in vitro with IL-1β and Pam-2-Cys, both known to induce expression of the pro inflammatory cytokines tumour necrosis factor-α and IL-8 via a nuclear factor-κB dependent pathway, resulted in the inhibition of HBV DNA replication in the transduced HepG2 cells by up to 90% and nucleocapsid formation in the transiently transfected Huh-7 cells by up to 30%, when compared with mock-treated cells. Conclusions Hepatoma cell lines expressed functional IL-1 receptor and TLR2 receptors, which when stimulated led to a signalling cascade that inhibited HBV replication. These data support an active role for hepatocytes in inhibiting HBV replication and provide a rationale for the development of TLR agonists as potentially novel antiviral agents.