In　hepatitis　B　virus　(HBV)　infection,　the　virus　produces　redundant　hepatitis　B　surface　antigen　(HBsAg)　that　plays　a　key　role　in　driving　T-cell　tolerance　and　viral　persistence.　However,　currently　available　anti-HBV　agents　have　no　direct　effect　on　HBsAg　transcription　and　protein　expression.　In　this　study,　we　designed　a　heat　shock　protein　gp96　inhibitor　p37　with　the　cell　penetrating　peptide　PTD　(protein　transduction　domain　of　trans-activator　of　transcription),　which　mediated　p37　internalization　into　hepatocytes.　PTD-p37　effectively　suppressed　HBsAg　expression　and　viral　replication　both　in　vitro　and　in　vivo.　We　further　provide　evidence　that　PTD-p37　suppressed　HBV　enhancer/promoter　activity　via　p53　upregulation.　Moreover,　PTD-p37　had　antiviral　activity　against　a　lamivudine-resistant　HBV　strain.　Considering　that　suppression　of　HBsAg　expression　is　a　major　goal　for　treatment　of　HBV　infection,　our　results　provide　a　basis　for　developing　a　new　therapeutic　approaches　targeting　host　factors　against　viral　expression.