AbstractBackgroundFolate is essential for normal foetal development as it plays an important role for gene expression during different periods of foetal development. Thus, prenatal exposure to folate may have a programming effect on pubertal timing.ObjectivesTo study the association between maternal intake of folate during pregnancy and pubertal timing in girls and boys.MethodsWe studied 6585 girls and 6326 boys from a Danish population‐based Puberty Cohort, 2000–2021. Information on maternal intake of folate from diet and folic acid from supplements was obtained from a food‐frequency questionnaire in mid‐pregnancy, and total folate was calculated as dietary folate equivalents. Information on age at menarche in girls, age at first ejaculation and voice break in boys, and Tanner stages, acne and axillary hair in both girls and boys was obtained every 6 months throughout puberty. We estimated mean monthly differences according to exposure groups for each pubertal milestone in addition to a combined estimate for the average age at attaining all pubertal milestones using multivariable interval‐censored regression models. Total folate was analysed in quintiles, continuous and as restricted cubic splines.ResultsMaternal intake of total folate in mid‐pregnancy was not associated with pubertal timing in girls (combined estimate for overall pubertal timing per standard deviation (SD 325 μg/day) decrease in maternal intake of total folate: −0.14 months (95% confidence interval [CI] −0.51, 0.22)). Boys had slightly later overall pubertal timing per standard deviation (SD 325 μg/day) decrease in maternal intake of total folate (combined estimate: 0.40 months, 95% CI 0.01, 0.72). Spline plots supported these findings.ConclusionsPrenatal exposure to low maternal intake of total folate in mid‐pregnancy was not associated with pubertal timing in girls but associated with slightly later pubertal timing in boys. This minor delay is likely not of clinical importance.