Mage-D1 (MAGE family member D1) is involved in a variety of cell biological effects. Recent studies have shown that Mage-D1 is closely related to tooth development, but its specific regulatory mechanism is unclear. The purpose of this study was to investigate the expression pattern of Mage-D1 in rat tooth germ development and its differential mineralization ability to ectomesenchymal stem cells (EMSCs), and to explore its potential mechanism. Results showed that the expression of Mage-D1 during rat tooth germ development was temporally and spatially specific. Mage-D1 promotes the proliferation ability of EMSCs but inhibits their migration ability. Under induction by mineralized culture medium, Mage-D1 promotes osteogenesis and tooth-forming ability. In vitro, Mage-D1 not only binds to p75 neurotrophin receptor (p75NTR) but also to distal-less homeobox 1(Dlx1) and msh homeobox 1 (Msx1). These findings indicate that Mage-D1 is absolutely important in tooth germ development. p75NTR, Dlx1, and Msx1 seem to be closely related to the underlying mechanism of Mage-D1 action.