© 2022. All Rights Reserved.Objective: Methotrexate (MTX) is an important chemotherapeutic and an important anticarcinogen used in cancer patients, but it causes side effects in other tissues. Among these side effects is lung toxicity. Vitamin B12 is a powerful antioxidant and reduces reactive oxygen species. This study was designed to determine whether vitamin B12 could protect against methotrexate-induced damage in rat lungs. Methods: A total of 32 male Wistar albino rats were divided into four groups: The control group (n=8) received intraperitoneal (i.p.) saline throughout the experiment. Vitamin B12 group (B12) (n=8) 3 µg/ kg/i.p. Vitamin B12 was administered for 14 days. The methotrexate (MTX) group received a single dose MTX injection at 20 mg/kg/i.p. on the 8th day of the experiment. On the 8th day of the experiment, a single dose of MTX 20 mg/kg/i.p. was given to the MTX+B12 group. + 3 µg/kg/i.p. Vitamin B12 was administered daily throughout the experiment. Histopathological, immunohistochemical, and biochemical methods were applied to the obtained lung tissues. Lung tissues were stained with Masson's Trichrome (MT). In addition, α-Sma, Laminin, PCNA, and TNF-α antibodies were stained by immunohistochemistry. Superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels were evaluated in lung tissue homogenates. Results: MTX caused an increase in MDA and IL-6 levels and expression of α-Sma, Laminin, and TNF-α, and the number of apoptotic cells in lung tissue. It also caused a decrease in PCNA expression and SOD and CAT levels in the MTX group. Vitamin B12 inhibited the increase in MDA and IL-6 levels and the expression of α-Sma, Laminin, and TNF-α. Vitamin B12 was found to increase antioxidant capacity. Conclusion: Vitamin B12 has been shown to be effective on factors such as oxidative stress, inflammation, fibrosis, and apoptosis in MTX-induced lung toxicity and reduce damage. We observed that MTX stimulated lung injury decreased with vitamin B12 treatment It suggests that vitamin B12 should not be ignored in reducing side effects in cancer drug use.