Hepatitis B virus (HBV) infection is a major health concern around the world, and only a minority of patients receiving current treatments achieve a sustained response following a short course of therapy. The majority of patients require prolonged therapy to suppress viral replication. However, several recent reports show that inhibiting host-cell proteins can prevent viral infection. Human epiregulin (EREG) was confirmed by our previous study to be up-regulated in HepG2.2.15 cells while down-regulated by lamivudine. Therefore, the question was asked whether inhibiting EREG could prevent viral infection. To address this question, antisense oligonucleiotides (ASODNs) were used to down-regulate EREG expression in HepG2.2.15 cells. We were able to show that HBV propagation in HepG2.2.15 cell culture was reduced in a dose-dependent manner by EREG inhibition. In addition, we found that treatment with ASODNs did not affect HepG2.2.15 cell viability. To probe the role of EREG in HBV replication, the interaction between EREG and HBsAg was also verified using co-immunoprecipitation and GST pull-down assays. We observed that EREG can contribute to HBsAg binding to HepG2 cells. In summary, this study demonstrates that EREG is essential for HBV replication and also provides some evidence for the potential role of EREG in HBsAg binding.