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Background: Inadequate vitamin D levels may increase the risk of caries during childhood. The purpose of this study was to investigate the association between 25-hydroxyvitamin D (25(OH)D) status and severe early childhood caries (S-ECC) in preschool children., Methods: Data were obtained from children <72 months of age in two case-control studies in Winnipeg, Manitoba and Richmond, Virginia. Serum analysis assessed 25(OH)D, calcium and parathyroid concentrations. Data on demographics, dental history and oral hygiene were obtained via questionnaires. Bivariate and multiple logistic regression analyses were performed to assess the relationships between demographic and biological variables and S-ECC. A p -value of ≤0.05 was significant., Results: Data were available for 200 children with S-ECC and 144 caries-free controls. Children with S-ECC had significantly lower 25(OH)D levels than those who were caries-free ( p < 0.001), and children with deficient 25(OH)D levels were 10 times more likely to have S-ECC ( p < 0.001). Multiple logistic regression revealed that having higher 25(OH)D and calcium concentrations ( p = 0.019 and p < 0.0001, respectively), as well as being breastfed in infancy ( p < 0.001), were significantly and independently associated with lower odds of S-ECC, while dental insurance ( p = 0.006) was associated with higher odds of S-ECC., Conclusions: This study provides additional evidence of an association between nutritional status, specifically vitamin D and calcium levels, and S-ECC.