Vitamin D has shown immune-modulatory effects but mostly in in vitro and animal studies. Regulatory T cells (Treg) are important for a balanced immune system. The relationship between vitamin D on the number of circulating neonatal Treg is unclear. We sought to investigate the association between maternal and neonatal vitamin D metabolites and cord blood (CB) Treg subsets. In a cohort of Australian infants (n = 1074), recruited using an unselected antenatal sampling frame, 158 mother–infant pairs had data on the following: 1) 25-hydroxyvitamin D₃ (25(OH)D₃) measures in both maternal peripheral blood (28- to 32-wk gestation) and infant CB; 2) proportions (percentage of CD4⁺ T cells) of CB Treg subsets (CD4⁺CD45RA⁺ FOXP3^low naive Treg, and CD4⁺CD45RA² FOXP3^high activated Treg [aTreg]); and 3) possible confounders, including maternal personal UV radiation. Multiple regression analyses were used. The median 25(OH)D₃ was 85.4 and 50.7 nmol/l for maternal and CB samples, respectively. Higher maternal 25(OH)D₃ levels were associated with increased CB naive Treg (relative adjusted mean difference [AMD] per 25 nmol/l increase: 5%; 95% confidence interval [CI]: 1–9%), and aTreg (AMD per 25 nmol/l increase: 17%; 95% CI: 6–28%). Furthermore, a positive association between CB 25(OH)D₃ levels and CB aTreg (AMD per 25 nmol/l increase: 29%; 95% CI: 13–48%) was also evident. These results persisted after adjustment for other factors such as maternal personal UV radiation and season of birth. 25(OH)D₃, may play a role in the adaptive neonatal immune system via induction of FOXP3⁺ Tregs. Further studies of immune priming actions of antenatal 25(OH)D₃ are warranted.