Hepatitis B virus (HBV) is one of the major causes of chronic liver diseases and hepatocellular carcinoma. In this study, we used a single chain antibody (sFv), which is a man-made antibody with a strong affinity of immunoglobulin, to inhibit HBV replication. Because HBV replication can only take place in the viral nucleocapsid made of HBV core protein (HBc), we generated anti-HBc sFv and examined whether intracellular anti-HBc sFv could inhibit viral replication in the human hepatoblastoma-derived cell line that produces HBV (HB611). With respect to HBV replication intermediates, both single-stranded and partially double-stranded DNA intermediates were markedly suppressed in the cells expressing anti-HBc sFv, although HBV RNA intermediates were not affected. This suggested that intracellular anti-HBc sFv inhibited HBV DNA replication by inhibiting reverse transcription from HBV pregenome RNA to single-stranded DNA. Because the sFv-HBc complex was detected in the cells expressing anti-HBc sFv by immunoprecipitation analysis but the quantity of intracellular HBc was not affected, the anti-HBc sFv was suggested to inhibit HBV DNA replication by interfering with the function of HBc. These results indicate that intracellular sFv against HBc might be effective as a novel active molecule for gene therapy of hepatitis B.