MSX1 mutations cause cleft lip/palate and hypodontia (Nat Genet, 1996). Therefore, elucidation of MSX1 actions in hard tissue differentiation of neural crest-derived mesenchymal cells is essential. MSX1 has been shown to suppress precocious hard tissue differentiation of dental mesenchymal cells at the early embryonic stage (Dev Biol, 2010). However, the role of MSX1 in development and regeneration of teeth has not been fully understood. We found that MSX1 knockdown abolished the induction of bone-related genes and calcification in dental pulp stem cell cultures. MSX1 knockdown also modulated the expression of Wnt, Notch and BMP signaling pathway genes. Unexpectedly, MSX1 knockdown increased the expression of sterol regulatory element-binding protein/SREBP in the osteogenesis-induction medium, along with many sterol synthesis-related genes, which are direct targets of the SREBP transcription factor. Furthermore, MSX1 knockdown enhanced adipogenic differentiation of dental pulp stem cells after exposure to adipogensis-induction medium. These findings suggest that MSX1 enhances hard tissue differentiation of dental pulp stem cells partly by the suppression of sterol metabolism and adipocyte differentiation.