Background and Aims: Change of gut microbiota com-position is associated with the outcome of hepatitis B virus (HBV) infection, yet the related mechanisms are not fully characterized. The objective of this study was to investigate the immune mechanism associated with HBV persistence induced by gut microbiota dysbiosis. Methods: C57BL/6 mice were sterilized for gut-microbiota by using an antibi-otic (ABX) mixture protocol, and were monitored for their serum endotoxin (lipopolysaccharide [LPS]) levels. An HBV-replicating mouse model was established by performing HBV-expressing plasmid pAAV/HBV1.2 hydrodynamic injec-tion (HDI) with or without LPS, and was monitored for se-rum hepatitis B surface antigen, hepatitis B e antigen, HBV DNA, and cytokine levels. Kupffer cells (KCs) were purified from antibiotic-treated mice and HBV-replicating mice and analyzed for IL-10 production and T cell suppression ability. Results: ABX treatment resulted in increased serum LPS levels in mice. The KCs separated from both ABX-treated and LPS-treated HBV-replicating mice showed significantly increased IL-10 production and enhanced ability to suppress IFN-γ production of TCR-activated T cells than the KCs sep-arated from their counterpart controls. HDI of pAAV/HBV1.2 in combination with LPS in mice led to a delayed HBV clear-ance and early elevation of serum IL-10 levels compared to pAAV/HBV1.2 HDI alone. Moreover, IL-10 function blockade or KC depletion led to accelerated HBV clearance in LPS-treated HBV-replicating mice. Conclusions: Our results suggest that dysbiosis of the gut microbiota in mice leads to endotoxemia, which induces KC IL-10 production and strengthens KC-mediated T cell suppression, and thus fa-cilitates HBV persistence.