The Australian Study of Causes of Acute Lymphoblastic Leukemia in Children (Aus-ALL) was designed to test the hypothesis, raised by a previous Western Australian study, that maternal folic acid supplementation during pregnancy might reduce the risk of childhood acute lymphoblastic leukemia (ALL). Aus-ALL was a national, population-based, multicenter case–control study that prospectively recruited 416 cases and 1,361 controls between 2003 and 2007. Detailed information was collected about maternal use of folic acid and other vitamin supplements before and during the index pregnancy. Data were analyzed using logistic regression, adjusting for matching factors and potential confounders. A meta-analysis with the results of previous studies of folic acid supplementation was also conducted. We found weak evidence of a protective effect of maternal folate supplementation before pregnancy against risk of childhood ALL, but no evidence for a protective effect of its use during pregnancy. A meta-analysis including this and 2 other studies, but not the study that raised the hypothesis, also found little evidence that folate supplementation during pregnancy protects against ALL: the summary odds ratios (ORs) for folate supplementation were 1.06 [95% confidence interval (CI): 0.77–1.48] with reference to no folate supplementation and 1.02 (95% CI: 0.86–1.20) with reference to no vitamin supplementation. For vitamin supplementation in general, the summary OR from a meta-analysis of 5 studies—including Aus-ALL—was 0.83 (95% CI: 0.73–0.94). Vitamin supplementation in pregnancy may protect against childhood ALL, but this effect is unlikely to be large or, if real, specifically due to folate.