Paeoniflorin (PF) is one of the main effective components of the total glucosides of peony, which has been reported to have anti-inflammatory ability. However, the effects of paeoniflorin on concanavalin A (Con A)-induced hepatitis have not been carefully examined. The aim of this study was to investigate the protective effect of paeoniflorin and elucidate potential mechanisms of paeoniflorin on Con A-induced hepatitis. C57BL/6 mice were divided randomly into the following four experimental groups: PBS group, PF group, Con A group, and Con A + PF group. Mice received paeoniflorin (50 mg/kg) by tail vein before Con A intravenous administration. We found that paeoniflorin pretreatment can significantly reduce the elevated plasma aminotransferase levels and liver necrosis in Con A-induced hepatitis. Also, paeoniflorin pretreatment suppressed the secretion of proinflammatory cytokines (TNF-α, INF-γ, IL-6), compared with Con A group. Meanwhile, paeoniflorin pretreatment decreased CD4+, CD8+ and NKT cell infiltration in the liver. Besides, we observed that paeoniflorin pretreatment can decrease the expression level of Toll-like receptor (TLR) 4 mRNA or protein in liver tissues. Further results showed that paeoniflorin pretreatment was capable of suppressing the activation of the NF-κB pathway by inhibiting IκBα kinase and p65 phosphorylation in Con A-induced liver injury. These results suggest that paeoniflorin pretreatment protects mice against Con A-induced liver injury via inhibition of several inflammatory mediators and, at least in part, by suppressing CD4+, CD8+ and NKT cell infiltration in liver. The beneficial effect of paeoniflorin may be related to the downregulation of TLR4 expression and the inhibition of NF-κB activation.