Methotrexate (MTX) is a broadly used anticancer. Its major side effect is hepatotoxicity. Gossypin is a flavonoid has a hepatoprotective effect as well as antitumor property. The study aimed at inspecting the protective effect of gossypin against MTX hepatotoxicity. Twenty-four adult male rats arranged into four groups (six rats each): control, gossypin control, MTX, and MTX+ gossypin. Animals were orally administered gossypin at 10 mg kg day for 7 days. MTX was injected i.p. (20 mg/kg once) on 5 day. Liver enzyme and oxidative stress markers were assessed. BAX, transforming growth factor-beta (TGF-β) gene expressions, and P-glycoprotein (P-gp) were assessed. The histopathological study as well as the immunohistochemical study for hepatic caspase 3 and nuclear factor kappa-B (NFκ-B) was done. MTX produced a significant increase of liver enzymes and distortion of hepatic architecture alongside with increased the hepatic collagen content. MTX administration significantly increased the oxidative stress markers and upregulated the pro-apoptotic BAX and the pro-fibrogenic TGF-β. MTX increased caspase 3 and NFκ-B expression, while diminished the expression of P-gp. Gossypin pretreatment improved the previous parameters, restored the normal hepatic architecture, reduced the hepatic fibrosis, and regained nearly normal expressions for BAX, TGF-β, caspase 3, and NFκ-B. Gossypin caused more reduction in P-gp hepatic expression. Gossypin may be a valuable adjuvant therapy that protects the liver against MTX toxicity through antioxidant, anti-inflammatory, antiapoptotic mechanisms, and mediated P-gp expression reduction.