This study aimed to investigate the effect of the timing, dose, and source of folate during pregnancy on childhood asthma by using data from an Australian prospective birth cohort study (n = 557) from 1998 to 2005. At 3.5 years and 5.5 years, 490 and 423 mothers and children participated in the study, respectively. Maternal folate intake from diet and supplements was assessed by food frequency questionnaire in early (<16 weeks) and late (30-34 weeks) pregnancy. The primary outcome was physician-diagnosed asthma, obtained by maternal-completed questionnaire. Asthma was reported in 11.6% of children at 3.5 years (n = 57) and in 11.8% of children at 5.5 years (n = 50). Folic acid taken in supplement form in late pregnancy was associated with an increased risk of childhood asthma at 3.5 years (relative risk (RR) = 1.26, 95% confidence interval (CI): 1.08, 1.43) and with persistent asthma (RR = 1.32, 95% CI: 1.03, 1.69). The effect sizes did not change with adjustment for potential confounders. The association was similar at 5.5 years but did not reach statistical significance (RR = 1.17, 95% CI: 0.96, 1.42) in univariable models. These findings on childhood asthma support previous observations that supplementation with folate in pregnancy leads to an allergic asthma phenotype in mice via epigenetic mechanisms and is associated with poorer respiratory outcomes in young children.