Childhood brain tumors (CBT) are the second most common childhood cancers, yet their etiology is largely unknown. We investigated whether maternal gestational intake of folate and vitamins B6 and B12 was associated with CBT risk in a nationwide case-control study conducted 2005-2010. Case children 0-14 years were recruited from all 10 Australian pediatric oncology centers. Control children were recruited by national random digit dialing, frequency matched to cases on age, sex, and state of residence. Dietary intake was ascertained using food frequency questionnaires and adjusted for total energy intake. Data from 293 case and 726 control mothers were analyzed using unconditional logistic regression. The odds ratio (OR) for the highest versus lowest tertile of folate intake was 0.70 [95% confidence interval (CI): 0.48, 1.02]. The ORs appeared lower in mothers who drank alcohol during pregnancy (OR = 0.45, 95% CI: 0.22, 0.93), mothers who took folic acid (OR = 0.67, 95% CI: 0.42, 1.06) or B6/B12 supplements (OR = 0.51, 95% CI: 0.25, 1.06) and in children younger than 5 years (OR = 0.50, 95% CI: 0.27, 0.93). These findings are consistent with folate's crucial role in maintenance of genomic integrity and DNA methylation. Dietary intake of B6 and B12 was not associated with risk of CBT.