Methotrexate (MTX) is a chemotherapeutic drug that is frequently used in the treatment of cancer. However, pulmonary toxicity is one of its serious life-threatening side effects. Our study aims to investigate the effect of rutin on MTX-induced pulmonary toxicity in rats biochemically and histopathologically. The experiment was performed on 18 albino Wistar rats, including the healthy group (C), MTX group (MTX), and MTX + routine group (MTX+R). As a result, rutin suppressed the increase of MDA and the decrease of tGSH with MTX administration. Rutin significantly decreased the amount of TNF-alpha, while NF-kappa B increased with MTX. Rutin also reduced the histopathological findings, such as severe chronic inflammatory cell infiltration in the peribronchial and perivascular areas, bronchial dilatation, pulmonary artery vessel wall thickening, pulmonary artery dilation, and congestion. Rutin attenuated MTX-induced oxidative and inflammatory lung injury. These findings indicate that rutin may be useful in the treatment of MTX-related lung injury.