Aim: The aim of this study was to assess biomarkers of calcium homeostasis and tooth development, in mothers during pregnancy and their children at birth, for enamel hypoplasia (EH) in the primary maxillary central incisor teeth. Methods: Bayesian methodology was used for secondary data analyses from a randomized, controlled trial of prenatal vitamin D<sub>3</sub> supplementation in healthy mothers (N = 350) and a follow-up study of a subset of the children. The biomarkers were serum calcium (Ca), phosphorus (P), intact parathyroid hormone (iPTH), total circulating 25-dihydroxyvitamin D (25(OH)D), and 1,25-dihydroxyvitamin D (1,25(OH)<sub>2</sub>D). The maternal biomarkers were assayed monthly during pregnancy, and the child’s biomarkers were derived from cord blood. Digital images of the child’s 2 teeth were scored for EH using Enamel Defects Index criteria for each of the incisal, middle, and cervical regions for an EH extent score. Results: The child EH prevalence was 41% (60/145), with most defects present in the incisal and middle tooth regions. Cord blood iPTH and 1,25(OH)<sub>2</sub>D levels were significantly associated with EH extent after controlling for maternal factors. For every 1 pg/mL increase in cord blood iPTH, the EH extent decreased by approximately 6%. For every 10 pg/mL increase in cord blood 1,25(OH)<sub>2</sub>D, the EH extent increased by almost 30% (holding all other terms constant and adjusting for subject-level heterogeneity). The relationship between maternal 25(OH)D and maternal mean iPTH varied significantly by EH extent. Conclusion: The results suggest possible modifiable relationships of maternal and neonatal factors of calcium homeostasis during pregnancy and at birth for EH, contributing to the frontier of knowledge regarding sound tooth development for dental caries prevention.