Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.