Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation, resulting in a hypomaturation AI phenotype, to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed that Wdr72−/− ameloblasts were shorter, and the enamel matrix was retained during maturation stage. H+/Cl− exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72−/− maturation-stage ameloblasts. Other markers along the endocytic degradative pathway were disrupted in Wdr72−/− mice, including clathrin, Dynamin II, and ANXA8. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72−/− mice as compared to wildtype and heterozygous controls. Moreover, Wdr72−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. In vivo and in vitro tracer studies with HRP and amelogenin showed delayed processing and loss of transport to lysosomes through the degradative pathway in our newly developed Wdr72−/− ameloblast-like cell line using CRISPR/Cas9. These cells enabled live cell mechanistic studies targeting WDR72 function in vesicle acidification and microtubule recruitment, revealing a complex relationship with a major vesicle acidifying protein, vacuolar H+-ATPase (v-ATPase), and a link to microtubule recruitment. Ultrastructure of Wdr72−/− ameloblasts also showed faulty vesicle formation and ruffled border formation that coincided with pH and calcium matrix defects. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins by regulating microtubule assembly and membrane turnover in the degradative pathway.