The elaborate modulation of the transforming growth factor β (TGF-β) superfamily signaling network plays an essential role in tooth morphogenesis and differentiation. In our previous studies, we have demonstrated that TGF-β1 promotes enamel mineralization and maturation using TGF-β1 gene conditional knockout (TGF-β1-cKO) mice. However, the specific regulatory mechanisms of TGF-β1 during enamel development remain unclear. Furthermore, we have previously found that the expression of WD repeat-containing protein 72(WDR72)in mouse enamel epithelium is decreased significantly in the absence of TGF-β1. Therefore, the aim of the present study was to investigate how TGF-β1 affects amelogenesis by regulating the expression of Wdr72. Histological examination showed that the absence of TGF-β1 in ameloblastic epithelial cells resulted in a reduction in enamel mineralization and a delay in enamel matrix protein absorption. TGF-β1, Runt-related transcription factor 2(RUNX2) and WDR72 were revealed to be colocalized in ameloblasts by immunohistochemistry, and it was also found that the expression of Runx2 and Wdr72 was markedly different between TGF-β1-cKO mice and wild type(TGF-β1-WT)mice. In addition, the effect of exogenous TGF-β1 on Wdr72 was more significant when RUNX2 was present than when RUNX2 was absent. Furthermore, we found that there were binding sites for RUNX2 on the promoter of Wdr72 and that Wdr72 expression was regulated by RUNX2. Collectively, our results suggest that TGF-β1 affects enamel mineralization by modulating RUNX2 and thus affecting the expression of Wdr72.