Hepatitis B virus (HBV) infection affects 364 million people worldwide and causes a serious global public health problem. The SRY-related high mobility group-box 9 (SOX9) is a risk of developing cirrhosis in patients with chronic hepatitis B and a cancer stem cell marker. However, the role of SOX9 in HBV replication has not been reported. This study revealed a distinct mechanism underling the regulation of HBV replication mediated by SOX9. HBV induces SOX9 mRNA and protein expression in human hepatoma cells, including HepG2.2.15, HepG2, Huh7, and HepG2-NTCP cells. Further study demonstrated that HBV activates SOX9 expression at the transcriptional level through inducing SOX9 promoter activity and HBc could induce the activity of SOX9 promoter. Interestingly, SOX9 in turn represses HBV replication in human hepatoma cells. More importantly, SOX9 inhibits HBV infection in HepG2-NTCP cells and C57/BL6 mice. Detailed study revealed that SOX9 suppresses HBV replication through directly binding to HBV EnhII/Cp (HBV 1667-1672 nt) to inhibit EnhII/Cp activation. Results from deletion mutant analysis, ChIP assay, nuclear and cytoplasmic extraction analysis, and immunofluorescence demonstrated that SOX9 high mobility group (HMG) domain is required for SOX9 anti-HBV activity. Moreover, we demonstrated that SOX9 and hepatocyte nuclear factor 4 alpha (HNF4α) can bind to HBV EnhII/Cp (HBV 1667-1672 nt) individually and simultaneously to regulate the promoter activity. Collectively, the results revealed a distinct negative feedback mechanism underlying HBV replication and SOX9 expression, and identified SOX9 as a new host restriction factor in HBV replication and infection. IMPORTANCE: HBV infection is a global public health problem by causing serious liver diseases, but the mechanisms underlying HBV pathogenesis remain largely unknown. SOX9 is a risk of developing cirrhosis and a cancer stem cell marker, however, the role of SOX9 in HBV infection has not been reported. The authors revealed a distinct mechanism underling the regulation of HBV replication and SOX9 expression. On the one hand, HBV induces SOX9 expression in human hepatoma cells through activating SOX9 promoter. On the other hand, SOX9 in turn represses HBV replication in human hepatoma cells by binding to and inhibiting HBV EnhII/Cp through its HMG domain. More importantly, SOX9 inhibits HBV infection in HepG2-NTCP cells and C57/BL6 mice. Therefore, this study identifies SOX9 as a novel and potential therapeutic reagent for the prevention and treatment of HBV-associated diseases.