Objective To explore the protective effect of hesperidin pretreatment on concanavalin A (Con A)-induced acute liver injury and the effect on expression of TNF-α and IFN-γ. Methods Seventy-two SPF C57BL/ 6 mice were randomly divided into three groups: normal control group, model control group and hesperidin group. Acute liver injury model was established by injected with Con A. The hesperidin group was treated intragastrically with 1 000 mg·kg-1 hesperidin for 10 days. Model control group was treated intragastrically with the same volume of 0. 5% of sodium carboxymethyl cellulose. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase ( AST) were measured. Pathological changes in hepatic tissue were observed under microscope. The expression of TNF-α and IFN-γ mRNAs in hepatic tissue was measured by reverse transcription polymerase chain reaction ( RT-PCR). The contents of TNF-α and IFN-γ in serum were detected by ELISA. Results Compared with model control group, the contents of ALT and AST in serum were significantly decreased (P<0. 01) in hesperidin group. Pathological changes in hepatic tissue were markedly improved. The expression of TNF-α and IFN-γ in the hepatic tissues and serum were significantly downregulated (P<0. 01). The concentrations of TNF-α and IFN-γ in hesperidin group were (717. 05±205. 22) and(611. 06±92. 82)pg·mL-1 in 2 h,(811. 56±167. 47)and(786. 19±215. 44)pg·mL-1 in 6 h. Compared with model control group, the expressions of TNF-α and IFN-γ in the hesperidin group were significantly downregulated (P<0. 01). But there was no significant difference between hesperidin group and model control group in 6 h after treated with Con A(P>0. 05). Conclusion Hesperidin pretreatment protects mice from Con A-induced acute liver injury possibly by inhibiting the expression of TNF-α and IFN-γ in the liver of mice.