Background: Whether antenatal and neonatal Vitamin D status have clinical relevance in fracture prevention has not been examined extensively, although observational studies indicate that fetal life may be a sensitive period in relation to bone growth and mineralization during childhood. Objective: We examined whether 25-hydroxyVitamin D3 [25(OH)D3] concentrations in stored neonatal dried blood spot (DBS) samples are associated with pediatric fracture risk. We hypothesized that in particular, low neonatal Vitamin D status may be a risk factor for fracture incidence among children. Design: In a register-based case-cohort study design, the case group was composed of 1039 individuals who were randomly selected from a total of 82,154 individuals who were born during 1989-1999 and admitted to a Danish hospital with a fracture of the forearm, wrist, scaphoid bone, clavicle, or ankle at age 6-13 y. The subcohort was composed of 1600 individuals randomly selected from all Danish children born during 1989-1999. The neonatal 25(OH)D3 concentrations in DBS samples were assessed by using highly sensitive chromatography-tandem mass spectrometry. Results: The mean ± SD 25(OH)D3 concentration for all subjects was 27.7 ± 18.9 nmol/L [median (IQR): 23.5 nmol/L (13.3, 37.3 nmol/L)] and showed significant monthly variation (P < 0.0001) with the highest values in July and August. Individuals in the middle quintile of neonatal 25(OH)D3 had lower odds of sustaining a fracture than did those in the lowest quintile (adjusted OR: 0.75; 95%CI: 0.58, 0.96), but a global test did not show any significant overall association (adjusted P = 0.13). Conclusions: This study suggested that neonatal Vitamin D status does not influence subsequent fracture risk in childhood. This is in accordance with studies that report no association between antenatal maternal Vitamin D status and childhood fractures. Further studies are needed to examine fracture risk in relation to prenatal Vitamin D status in a randomized controlled setting.