Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130Cas^Δepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130Cas^Δepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130Cas^Δepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130Cas^Δepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca²⁺+K⁺-exchanger in the maturation stage were reduced in p130Cas^Δepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis.