The liver is considered as a unique lymphoid organ favoring the induction of immune tolerance, rather than immunity. Biologists and clinicians alike have a long-standing interest in how the liver induces systemic immune tolerance, but the mechanism has not yet been well elucidated. Here, we employed hepatitis B virus (HBV)-carrier mice generated by hydrodynamically injecting phosphor-adeno-associated virus/HBV1.2 plasmid as a model for adult chronic HBV infection, which we found were unable to respond to hepatitis B surface antigen vaccination. Humoral tolerance induced in HBV-carrier mice could be transferred into Rag1−/− mice, because anti-HBV immunity in immunologically reconstituted Rag1−/− mice was inhibited by adoptive transfer of splenocytes from HBV-carrier mice. Humoral tolerance needed at least 7 days for induction and persisted to 3 months after a single HBV plasmid injection. Kupffer cell (KC) depletion or interleukin (IL-10) deficiency broke this humoral tolerance, and exogenous injection of IL-10 could effectively induce this tolerance. Conclusion: KCs in HBV-carrier mice expressed more IL-10 and mediated the systemic tolerance induction in an IL-10-dependent manner. This previously undescribed humoral tolerance regarding HBV infection will help to explore new approaches to reverse liver-sustained systemic immune tolerance in liver disease. (Hepatology 2014;59:443-452)