Methotrexate is a dihydrofolate reductase inhibitor widely employed in curative treatment for children with acute lymphoblastic leukemia (ALL). However, methotrexate administration is also associated with persistent cognitive deficits among long-term childhood cancer survivors. Animal models of methotrexate-induced cognitive deficits have primarily utilized adult animals. The purpose of present study is to investigate the neurotoxicity of methotrexate in juvenile rats and its relevant mechanisms. The doses and schedule of systemic and intrathecal methotrexate, given from post-natal age 3–7 weeks, were chosen to model the effects of repeated methotrexate dosing on the developing brains of young children with ALL. This methotrexate regimen had no visible acute toxicity and no effect on growth. At 15 weeks of age (8 weeks after the last methotrexate dose) both spatial pattern memory and visual recognition memory were impaired. In addition, methotrexate-treated animals demonstrated impaired performance in the set-shifting assay, indicating decreased cognitive flexibility. Histopathological analysis demonstrated decreased cell proliferation in methotrexate-treated animals compared to controls, as well as changes in length and thickness of the corpus callosum. Moreover, methotrexate suppressed microglia activation and RANTES production. In conclusion, our study demonstrated that a clinically relevant regimen of systemic and intrathecal methotrexate induces persistent deficits in spatial pattern memory, visual recognition memory and executive function, lasting at least 8 weeks after the last injection. The mechanisms behind methotrexate-induced deficits are likely multifactorial and may relate to suppression of neurogenesis, alterations in neuroinflammation and microglial activation, and structural changes in the corpus callosum.