Intrahepatic immune tolerance is critical to prevent potentially harmful inflammatory responses. Tr1 regulatory T cell (Tr1 cell) that mainly produces interleukin 10 (IL-10) is an efficient regulator in immune tolerance. However, whether Tr1 regulatory T cells also participate in intrahepatic immune tolerance is not clear yet. In this study, we found that a single injection of a sublethal dose of concanavalin A (ConA) into BALB/c mice induced tolerance against ConA-induced liver damage within 8 days. Further, we found that ConA pretreatment elicited high frequency of Tr1 cells characterized as a higher secretion of IL-10, a lower production of IL-4 and no expression of Foxp3. Importantly, adoptive transfer of Tr1 cells into naïve mice remarkably reduced ConA-mediated liver injury. The exerted inhibition function was through IL-10-dependent pathway. Treatment with IL-10 neutralized antibody in vivo significantly blocked the protective effect of IL-10 in ConA/ConA mice. Taken together, these results demonstrated that Tr1 cells elicited by ConA pretreatment played an important role in inducing immune tolerance in the liver. Our finding provided new insights into the involvement of Tr1 regulatory T cells in preventing liver from immune-mediated injury.