The signals driving T cell activation in T cell-mediated fulminant hepatitis are not fully understood. In this study, we identify the cytokine IL-27p28/EBI3 as a major pathogenic factor in the ConA model of T cell-mediated hepatitis. We found an up-regulation of hepatic EBI3 and p28 expression and augmented levels of IL-27 in wild-type mice after ConA administration, suggesting a potential pathogenic role of this cytokine in ConA hepatitis. Consistently, IL-27 EBI3-deficient mice were almost completely protected from ConA-induced liver damage. Such protection was associated with reduced levels of IFN-γ and its signaling proteins pSTAT-1 and T-bet. Finally, in vivo blockade of IL-27 function using a soluble IL-27 receptor fusion protein led to reduced pSTAT1 levels and suppression of liver injury. Taken together, these data demonstrate a key pathogenic role of IL-27 in T cell-mediated liver injury. Furthermore, in vivo blockade of IL-27 emerges as a novel potential therapy for T cell-mediated hepatitis.