Isoniazid (INH) and rifampicin (RFP), two front‐line drugs used in tuberculosis therapy, may lead to seriously hepatotoxicity. The current study was carried out to investigate the hepatoprotective effects of hesperidin against INH‐ and RFP‐induced oxidative damage. The liver injury animal model of rats was induced by INH (75 mg/kg) and RFP (150 mg/kg) coadministration for 7 days, and hesperidin, at the dose of 50, 100, and 200 mg/kg, was orally administered to rats 2 h before INH and RFP administration. The biochemical and pathologic examinations were performed after rats were sacrificed. Moreover, the serum and liver glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde (MDA), GSH peroxidase, and GSSG reductase were determined by test kits, and the expression of multidrug resistance proteins 2 (Mrp2) was determined by Western blotting and immunohistochemistry. The results showed that hesperidin significantly alleviated liver injury as indicated by the decreased levels of ALT, AST, bilirubin, total bile acid, and glutathione peroxidase and the increased levels of the GSH/GSSG ratio and the expression of Mrp2. Moreover, hesperidin could effectively reduce the pathological tissue damage. These results indicate that hesperidin can attenuate INH‐ and RFP‐induced oxidative damage, and the underlying mechanism may have correlation with its effect on the upregulation of Mrp2.