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)