Background <p>Reduced folate carrier 1 (RFC1) is an important solute carrier transporter crucial for the uptake and transport of methotrexate (MTX). This meta-analysis aimed to systemically analyze the relationship between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric acute lymphoblastic leukemia (ALL) patients.</p> Methods <p>A systematic search for studies reporting the correlation between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric ALL patients was performed via the Web of Science, EMBASE, PubMed, Wan Fang, CNKI, and VIP databases until June 16, 2025, followed by pooled analysis. The Newcastle‒Ottawa scale, Egger’s test, and leave‒one-out sensitivity analysis were performed to assess the study quality, publication bias and stability of the pooled results.</p> Results <p>Thirteen eligible studies were included in this meta-analysis, with 2, 5, and 6 studies having Newcastle‒Ottawa Scale scores of 7 points, 8 points, and 9 points, respectively, indicating good study quality. The pooled prevalence of the RFC1 80 AA genotype was 28.29% (95% CI: 23.44%-33.15%) in pediatric ALL patients. The risks of overall toxicity (OR = 4.68, 95% CI: 1.96–11.15), myelosuppression (OR = 1.85, 95% CI: 1.30–2.65), hepatotoxicity (OR = 2.44, 95% CI: 1.14–5.21), and gastrointestinal toxicity (OR = 1.61, 95% CI: 1.03–2.52) were greater in pediatric ALL patients with the RFC1 80 AA genotype than in those with the RFC1 80 GG + GA genotype who underwent MTX treatment. However, neurotoxicity risk (OR = 0.98, 95% CI: 0.40–2.41) and mucositis risk (OR = 1.24, 95% CI: 0.81–1.89) were not different between the two groups. No publication bias existed, while some of the pooled results were not stable according to the sensitivity analysis.</p> Conclusion <p>The RFC1 G80A polymorphism is closely correlated with elevated MTX-induced toxicity in pediatric ALL patients.</p>