The liver appears to play an important role in immunological tolerance, for example, during allo-transplantation. We investigated tolerance mechanisms in the model of concanavalin A (ConA)-induced immune-mediated liver injury in mice. We found that a single injection of a sublethal ConA dose to C57BL/6 mice induced tolerance toward ConA-induced liver damage within 8 days. This tolerogenic state was characterized by suppression of the typical Th1 response in this model and increased IL-10 production. Tolerance induction was fully reversible in IL-10−/− mice and after blockade of IL-10 responses by anti-IL10R antibody. Co-cultures of CD4+CD25+ regulatory T cells (Tregs) and CD4+CD25 responder cells revealed Treg from ConA-tolerant mice being more effective in suppressing polyclonal T cell responses than Treg from control mice. Moreover, Treg from tolerant but not from control mice were able to augment in vitro IL-10 expression. Depletion by anti-CD25 monoclonal antibody (MAb) indicated a functional role of Tregs in ConA tolerance in vivo. Cell depletion studies revealed Tregs and Kupffer cells (KC) to be crucial for IL-10 expression in ConA tolerance. Studies with CD1d−/− mice lacking natural killer T (NKT) cells disclosed these cells as irrelevant for the tolerogenic effect. Finally, cellular immune therapy with CD4+CD25+ cells prevented ConA-induced liver injury, with higher protection by Treg from ConA-tolerized mice. Conclusion: The immunosuppressive cytokine IL-10 is crucial for tolerance induction in ConA hepatitis and is mainly expressed by CD4+CD25+ Treg and KC. Moreover, Tregs exhibit therapeutic potential against immune-mediated liver injury. (HEPATOLOGY 2007;45:475–485.)