Background: Methotrexate, folic acid antagonist, is a widely used anticancer drug. Many cancers and inflammatory illnesses are treated with it because of its antiproliferative properties. Despite its therapeutic benefits, it produces toxic effects on various organs. Lung toxicity is a potential side effect of methotrexate. Thymoquinone is an oil extract of nigella sativa plant exhibiting great usage nowadays in medical field and attracting researchers due to its antioxidant, anticancer and anti-inflammatory characteristics. Aim of study: To investigate the potential protective role of thymoquinone against methotrexate-induced lung toxicity. Material and methods: sixteen rats were divided randomly into 4 groups (4 rats per group). GroupI received no treatment. GroupII received single dose methotrexate (20 mg/kg). GroupIII and GroupIV received thymoquinone 5 and 10 mg/kg/day for 11 days respectively. Biochemical parameters were done including Malondialdehyde, total glutathion, total antioxidant capacity, superoxide dismutase, and tumor necrosis factor α. Histopathological examinations with Haematoxylin & Eosin and Masson´s trichome stains were done. Results: In comparison to other groups, GroupII's exhibited significantly lower levels of tGSH, TAC, and SOD and significantly higher levels of MDA and TNFα. Groups III and IV exhibited significantly higher levels of SOD, tGSH, and TAC and significantly lower levels of MDA and TNFα, in comparison to groupII. Group II's hematoxylin and eosin sections revealed peribronchial lymphocytic aggregation, thicker alveolar walls, and congested capillaries. Groups III and IV's lung histology was almost normal. GroupII displayed peribronchial fibrosis shown by Masson's trichome stain, whereas GroupIII and GroupIV displayed minor interstitial tissue fibrosis.