Dental enamel formation depends upon the transcellular transport of Ca²⁺ by ameloblasts, but little is known about the molecular mechanism, or even if the same process is operative during the secretory and maturation stages of amelogenesis. Identifying mutations in genes involved in Ca²⁺ homeostasis that cause inherited enamel defects can provide insights into the molecular participants and potential mechanisms of Ca²⁺ handling by ameloblasts. Stromal Interaction Molecule 1 (STIM1) is an ER transmembrane protein that activates membrane-specific Ca²⁺ influx in response to the depletion of ER Ca²⁺ stores. Solute carrier family 24, member 4 (SLC24A4), is a Na⁺/K⁺/Ca²⁺ transporter that exchanges intracellular Ca²⁺ and K⁺ for extracellular Na⁺. We identified a proband with syndromic hypomaturation enamel defects caused by a homozygous C to T transition (g.232598C>T c.1276C>T p.Arg426Cys) in STIM1, and a proband with isolated hypomaturation enamel defects caused by a homozygous C to T transition (g.124552C>T; c.437C>T; p.Ala146Val) in SLC24A4. Immunohistochemistry of developing mouse molars and incisors showed positive STIM1 and SLC24A4 signal specifically in maturation-stage ameloblasts. We conclude that enamel maturation is dependent upon STIM1 and SLC24A4 function, and that there are important differences in the Ca²⁺ transcellular transport systems used by secretory- and maturation-stage ameloblasts.