Transforming growth factor β1 (TGF-β1) and Runt-related transcription factor 2 (RUNX2) are critical factors promoting enamel development and maturation. Our previous studies reported that absence of TGF-β1 or RUNX2 resulted in abnormal secretion and absorption of enamel matrix proteins. However, the mechanism remained enigmatic. In this study, TGF-β1Runx2 and TGF-β1Runx2 mice were successfully generated to clarify the relationship between TGF-β1 and RUNX2 during amelogenesis. Lower mineralization was observed in TGF-β1Runx2 and TGF-β1Runx2 mice than single gene deficient mice. Micro-computed tomography (μCT) revealed a lower ratio of enamel to dentin density in TGF-β1Runx2 mice. Although μCT elucidated a relatively constant enamel thickness, variation was identified by scanning electron microscopy, which revealed that TGF-β1Runx2 mice were more vulnerable to acid etching with lower degree of enamel mineralization. Furthermore, the double gene knock-out mice exhibited more serious enamel dysplasia than the single gene deficient mice. Hematoxylin-eosin staining revealed abnormalities in ameloblast morphology and arrangement in TGF-β1Runx2 mice, which was accompanied by the absence of atypical basal lamina (BL) and the ectopic of enamel matrix. Odontogenesis-associated phosphoprotein (ODAPH) has been identified as a component of an atypical BL. The protein and mRNA expression of ODAPH were down-regulated. In summary, TGF-β1 and RUNX2 might synergistically regulate enamel mineralization through the downstream target gene Odaph. However, the specific mechanism by which TGF-β1 and RUNX2 promote mineralization remains to be further studied.