BACKGROUND: Methotrexate (MTX) is an antifolate medication indicated to treat an array of tumors and autoinflammatory maladies. MTX may exhibit harmful impacts on multiple organs, especially liver injury and cirrhosis. Juniperus macrocarpa is a medicinal herb enriched with polyphenols and flavonoids featuring robust anti-inflammatory and antioxidative benefits. OBJECTIVE: To evaluate the hepatoprotective effects of Juniperus macrocarpa aqueous extract on MTX-aggravated liver toxicity. METHODS: The study involved 20 male middle-aged albino rats, arbitrarily allocated into 4 groups of 5 animals each. Group 1 (control) were given distilled water (DW) once daily for two weeks. Group 2 (MTX) got an intraperitoneal single dose of MTX (20 mg/kg) for two weeks. Rats in groups 3 and 4 were given daily dosages of 100 mg and 200 mg of Juniperus macrocarpa aqueous extract, respectively, for two weeks before receiving a single intraperitoneal MTX injection. RESULTS: Juniperus macrocarpa extracts at both low and high doses substantially alleviated the MTX-provoked biochemical alterations, as evidenced by decreased levels of inflammatory parameters including TNF-α and IL-6 and hepatic enzymes including ALT, AST, and ALP. Juniperus macrocarpa also significantly boosted levels of the anti-oxidant enzymes like SOD and GPX. Moreover, Juniperus macrocarpa extract attenuated congestive and degenerative hepatic changes, as indicated by improved histopathological findings. CONCLUSION: The anti-oxidative and anti-inflammatory activities of Juniperus macrocarpa extract are a promising approach for ameliorating MTX-aggravated hepatotoxicity.