The roles of interferon regulatory element (IRE) in Hepatitis B virus (HBV) genome on inhibitory effect of interferon against HBV are controversial in vitro. This study aimed to determine the functional characterization of HBV-IRE sequence in vivo. Wild-type or IRE-mutant HBV replication-competent mice were firstly established, and mice were subquently treated with polyinosinic-polytidylin acid (polyI.C) or phosphate-buffered saline via intraperitoneal. Results showed that PolyI.C inhibited viral replication, and increased the level of 2 0 ,5 0 -oligoadenylate synthase mRNA transcripts, a marker of INF-a/b induction. Between wildtype and IRE-mutant HBV replication-competent mice, the levels of HBV-RNA and HBV-DNA replication intermediates were similar. After PolyI.C treatment, the decreasing of HBV-RNA was similar between two groups, but HBV-DNA replication intermediates decreased significantly less in IREmutant than wild-type HBV replication-competent mice. These findings suggested that IRE mutant reduced the inhibitory effect of interferon on HBV replication, which played a role in antiviral effect of interferon against HBV.