Objective(s): Methotrexate (MTX) is a widely used chemotherapeutic agent that, however, is known to have serious side effects such as neurotoxicity. In the present study, we aimed to evaluate the possible favorable effects of ramelteon (RMLT) on MTX-induced cerebral toxicity.Materials and Methods: Thirty-two male Wistar albino rats were divided into four groups: Control group, MTX group (20 mg/kg MTX, IP, single dose), MTX+RMLT group (20 mg/kg MTX, IP, single dose + 10 mg/kg RMLT, by gavage, 7 days), and RMLT group (10 mg/kg RMLT, by gavage, 7 days).Results: In the MTX group, increased levels of total oxidant status (TOS) and oxidative stress index (OSI) levels and decreased levels of total antioxidant status (TAS) level were observed. RMLT significantly reversed oxidative stress parameters. Real-time PCR analysis revealed that MTX increased the expressions of Beclin-1 and autophagy-related gene 12 (ATG12). These expressions were significantly decreased by RMLT. Vacuolar changes, apoptotic cells, and inflammatory cell infiltration induced by MTX were ameliorated by RMLT treatment. Increased tumor necrosis factor-alpha (TNF-alpha) and Caspase-3 activities induced by MTX were returned to their normal levels by RMLT.Conclusion: All our results demonstrate that RMLT alleviates the harmful effects of MTX on the cerebral cortex tissue. Therefore, RMLT may be considered for supportive therapy for preventing side effects of MTX in patients needing MTX therapy.