Vitamin D deficiency has been associated with significant changes in dental structures. In children, it can induce enamel and dentin defects, which have been identified as risk factors for caries. This study aimed to assess the association between low serum 25-hydroxyvitamin D (25(OH) D) levels (<30 ng/mL) and the prevalence of caries in the permanent teeth and mixed dentition of 7-year-old children. A sample of 335 children from the population-based birth cohort Generation XXI (Porto, Portugal) was included. Data on children’s demographic and social conditions, health status, dental health behaviours, dental examination including erupted permanent first molars, and blood samples available for vitamin D analysis were collected. Dental outcomes included the presence of caries, including non-cavitated lesions (d1–6mft/D1–6MFT > 0), and advanced caries (d3–6mft/D3–6MF > 0). Serum 25(OH) D was measured using a competitive electrochemiluminescence immunoassay protein-binding assay. Bivariate analysis and multivariate logistic regression were used. Advanced caries in permanent teeth was significantly associated with children’s vitamin D levels <30 ng/mL, gastrointestinal disorders, higher daily intake of cariogenic food, and having had a dental appointment at ≤7 years old. Optimal childhood levels of vitamin D may be considered an additional preventive measure for dental caries in the permanent dentition.