As a common chemotherapy drug, methotrexate (MTX) has achieved remarkable clinical success. However, high inter-individual variability and unpredictable toxicity continue to challenge its use in clinical practices. Some studies suggest this variation is associated with a methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, but results remain unclear. In this meta-analysis, we include 14 studies that focus on MTHFR C677T and A1298C polymorphisms in pediatric patients with malignancy. We found significant associations of the MTHFR C677T polymorphism with hepatotoxicity (grade ≥2; CC vs CT/TT: risk ratio (RR): 0.82, 95% confidence interval (CI): 0.67-0.99; P = 0.04), hematological toxicity (grade 3-4; CC vs CT/TT: RR: 0.65, 95% CI: 0.44-0.97; P = 0.03) in a dominant genetic model and mucositis (grade ≥ 3) in all models (CC vs CT/TT: RR: 0.18, 95% CI: 0.04-0.87; P = 0.03; CC/CT vs TT: RR: 0.10, 95% CI: 0.03-0.32; P ≤ 0.0001; CC vs TT: RR: 0.10, 95% CI: 0.02-0.50; P = 0.005). No significant association was found with the MTHFR A1298C polymorphism. For children with malignancy, genotyping of the MTHFR C677T polymorphism is expected to be a useful tool in reducing toxicity and improving outcome in personalized MTX therapy.