Background and Objective:  While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells.Material and Methods:  To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a β‐galactosidase transgene under the control of a LEF/TCF and β‐catenin‐inducible promoter. To investigate in greater detail the molecular mechanisms involved in the β‐catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full‐length recombinant mouse amelogenin and were evaluated for phenotypic changes and β‐catenin signaling using a TOPFLASH construct and the LacZ reporter gene.Results:  In these in vitro models, we showed that amelogenin can activate β‐catenin signaling.Conclusion:  Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.