Odontomas are classified as odontogenic benign tumors, which show disorganized dental mineralized hard tissue formation in the jaw, but mechanisms in the induction of odontomas remain to be clarified. Odontomas are also thought to be developmental anomalies of tooth germ, which frequently occur in patients with familial adenomatous polyposis (FAP) involving activation of Wnt/b-catenin signaling. However, the roles of Wnt/b-catenin signaling in odontomas or odontogenic cells remain to be clarified. The current study was conducted to investigate the expression of b-catenin in odontomas and the function of Wnt/b-catenin signaling in odontogenic epithelial cells and tooth germ development. b-catenin frequently accumulated in the nucleus and/or cellular cytoplasm of remaining odontogenic epithelial cells in human odontoma specimens, immunohistochemically. Activation of Wnt/b-catenin signaling inhibited odontogenic epithelial cell proliferation and mouse tooth germ development, while inducing epithelial bud formation in the novel developed epithelial tooth bud culture system derived from mouse tooth germ. We identified Semaphorin 3A (Sema3A) as a downstream molecule of Wnt/b-catenin signaling using DNA microarray analysis and showed that Wnt/b-catenin signaling-dependent reduction of Sema3A expression resulted in suppression of odontogenic epithelial cell proliferation. Novel developed epithelial tooth bud culture system revealed that Sema3A expression is required in epithelial budding morphogenesis. These results suggest that Wnt/b-catenin signaling negatively regulates odontogenic epithelial cell proliferation and tooth germ development through decreased-Sema3A expression, and aberrant activation of Wnt/b-catenin signaling may be associated with the formation of odontomas. Overall design: DNA microarray analysis of mDE6 cells with 6 h stimulation of CHIR99021 was performed. Candidate genes were selected based on the criterion that their expression levels were lower or higher in cells treated with CHIR99021 than in the control cells.