Objectives: Inadequate maternal vitamin D (25(OH)D) levels during pregnancy may affect primary tooth calcification predisposing enamel hypoplasia (EH), a risk factor for Early Childhood Caries (ECC). The purpose of the study was to determine the relationship between 25(OH)D status of expectant mothers and the incidence of EH and ECC among their infants. Methods: This prospective study recruited expectant mothers during their second trimester of pregnancy. A prenatal questionnaire was completed and serum sample drawn for a 25(OH)D assay. Infant dental exams, scheduled at approximately 12 months of age, determined EH and ECC, at which time the mother completed a second questionnaire. The dental examiner was blinded to each mother's prenatal vitamin D status. Results: 207 women, 90% of whom self-declared Aboriginal heritage, were enrolled at a mean age of 19.0 ± 4.7 years. The mean serum 25(OH)D was 48.1 ± 24.4 nmol/L. 35% had levels ≤ 35 nmol/L, a formerly-defined threshold of deficiency. Only 10% of women had concentrations ≥ 80 nmol/L, denoting adequacy. 135 infants were examined at 16.1 ± 7.4 months of age. EH was identified in 22% of infants, 23% had ECC and (36% ECC when white spot lesions were included). Mothers of children with EH had lower mean serum 25(OH)D concentrations during pregnancy than mothers of children without EH (43.2 ± 21.1 vs. 51.4 ± 27.4 nmol/L, p=.072). Mothers of children with ECC had lower 25(OH)D levels than mothers whose children were caries-free (41.4 ± 20.4 vs. 52.4 ± 27.4 nmol/L, p=.045). The rate of untreated decay was inversely related with maternal vitamin D concentrations (p<.001). Infants with EH were significantly more likely to have ECC (p<.001). Logistic regression identified low maternal calcium levels (p=.034), not having heard of vitamin D (p=.036), and not using margarine daily (p=.024) as being significantly associated with EH in the primary dentition of infants. Backwards logistic regression revealed that EH (p<.001), infant age (p=.002), and lower 25(OH)D levels during pregnancy (p=.019) were significantly associated with ECC. Conclusions: This study shows for the first time that maternal vitamin-D levels may influence primary dentition and the development of ECC in their babies.