Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the stable genomic form as the template for viral transcription, plays a crucial role in viral persistence which remains a major global health problem. While accumulating evidence suggests the involvement of transcription factors and epigenetic machinery in cccDNA transcription, the roles of host transcription factors which contribute to epigenetic modification of cccDNA remain largely unknown. Zinc finger and homeoboxes 2 (ZHX2) is abundantly expressed in adult hepatocytes, where it acts as a transcriptional repressor and tumor suppressor by directly inhibiting the promoter activities of target genes. However, whether ZHX2 influences HBV replication or is involved in cccDNA epigenetic regulation remain unknown. In this study, we investigated the role of ZHX2 in cccDNA transcription. Analysis of immunohistochemistry showed that ZHX2 nuclear expression negatively correlated with serum HBV DNA and HBeAg. Remarkably, ZHX2 significantly decreased HBV antigens expression, pregenomic RNA (pgRNA) and HBV core particle DNA production both in vitro and in mouse livers supporting HBV antigens expression and cccDNA transcription. Dual luciferase and cccDNA ChIP assays confirmed that ZHX2 could bind to cccDNA and transcriptionally inhibit HBV promoter activities. In addition, ZHX2 suppressed the expression of histone regulator genes, such as cccDNA bound p300/CBP, and led to epigenetic repression of cccDNA. These findings highlight the roles of a novel restriction factor, ZHX2, in modulating HBV replication via regulating HBV promoter activities and cccDNA modifications. This study furthers our understanding of HBV transcription from cccDNA and offers new insights on potential HBV therapy.