Dentin is a major mineralized component of teeth. Odontoblasts are responsible for synthesis and secretion of dentin matrix. Previously, it has been demonstrated in a cell culture system that the E3 ubiquitin ligase, murine double minute 2 (Mdm2), promotes odontoblast-like differentiation of mouse dental papilla cells (mDPCs) by ubiquitinating p53 and the odontoblast-specific substrate Dlx3. However, whether Mdm2 plays an essential role in vivo in odontoblast differentiation and dentin formation remains unknown. In this study, we investigated the in vivo functions of Mdm2 using mice combined with multiple histological and molecular biological methods. The results showed that Mdm2 deletion in the odontoblast layer led to defects in odontoblast differentiation and dentin formation. Unexpectedly, specific inhibition of the Mdm2-p53 axis in wild-type mice by injection of a small-molecule inhibitor Nutlin-3a indicated that the role of Mdm2 in dentinogenesis was p53 independent, which was inconsistent with the previous in vitro study. In situ proximity ligation assay (PLA) showed that Mdm2 interacted with and ubiquitinated Dlx3 in the odontoblast nucleus of mouse molars. Dlx3 promoted the translocation of Mdm2 to the nucleus, and in turn, the nuclear Mdm2 mediated ubiquitination of Dlx3 and promoted the odontoblast-like differentiation of mDPCs. Dlx3 interacted with Mdm2 through its C-terminal domain. Deletion of the C-terminal domain of Dlx3 reversed the enhanced odontoblast-like differentiation and the activation of promoter mediated by overexpression of wild-type or nuclear Mdm2. Our findings suggest that nuclear Mdm2 mediates ubiquitination of the transcription factor Dlx3, which is essential for Dlx3 transcriptional activity on as well as subsequent odontoblast differentiation and dentin formation.