Background: Interest in a possible protective effect of maternal vitamin use before or during pregnancy against childhood brain tumors (CBT) and other childhood cancers has grown over the past decade. Our Australian study of CBTs, conducted between 2005 and 2011, investigated whether maternal use folic acid and other supplements was protective.Methods: Case children were identified through the 10 Australian pediatric oncology centers and controls were recruited by national random digit dialing. Mothers of 327 cases and 867 control children provided information on supplement use before and during the index pregnancy, including brand name, dose, and timing. Data were analyzed using multivariable unconditional logistic regression.Results: The OR for any maternal use of folic acid, use of folic acid without iron or vitamins B6, B12, C, or A, and any vitamin use before pregnancy, were: 0.68 [95% confidence interval (CI), 0.46–1.00; 0.55 (95% CI, 0.32–0.93) and 0.68 (95% CI, 0.46–1.01), respectively. The ORs for use of these supplements during pregnancy were also below unity, but generally closer to the null than those for the prepregnancy period. There was some evidence of an inverse dose–response during each time period.Conclusions: These results suggest that folic acid supplements before and possibly during pregnancy may protect against CBT. Such associations are biologically plausible through established mechanisms.Impact: This study provides evidence of a specific protective effect of prenatal folic acid supplementation against the risk of CBT that is not attributable to the actions of the other micronutrients investigated. ©2012 AACR.