Postnatally, severe vitamin D deficiency commonly results in rickets as well as potential defects in tooth mineralization. The effects of milder deficiency on oral health outcomes later in life are still unclear. This study used micro–computed tomography (μCT), energy dispersive X-ray analysis (EDX), and Raman spectroscopy to investigate mineral density, total density, and elemental composition of enamel and dentine in 63 exfoliated primary incisors from participants with known 25-hydroxyvitamin D levels (25-OHD) at birth. No differences in mineralization and chemical composition using μCT and EDX analysis were observed irrespective of 25-OHD status. Subtle structural differences were observed via Raman spectroscopy, with more crystalline enamel observed in those with sufficient 25-OHD at birth. Although subtle, the differences seen suggest further attention should be given to children with known milder levels of vitamin D deficiency in early life.