Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein found in dental and skeletal tissues. Although information regarding the role of MEPE in bone and disorders of phosphate metabolism is emerging, the role of MEPE in dental tissues remains unclear. We performed RNA in situ hybridization and immunohistochemistry analyses to delineate the expression pattern of MEPE during embryonic and postnatal development in craniofacial mineralizing tissues. Mepe RNA expression was seen within teeth from cap through root formation in association with odontoblasts and cellular cementoblasts. More intense expression was seen in the alveolar bone within the osteoblasts and osteocytes. MEPE immunohistochemistry showed biphasic dentin staining in incisors and more intense staining in alveolar bone matrix and in forming cartilage. Analysis of Mepe null mouse molars showed overall mineralized tooth volume and density of enamel and dentin comparable with that of wild-type samples. However, Mepe-/- molars exhibited increased thickness of predentin, dentin, and enamel over controls and decreased gene expression of Enam, Bsp, Dmp1, Dspp, and Opn by RT-PCR. In vitro Mepe overexpression in odontoblasts led to significant reductions in Dspp reporter activity. These data suggest MEPE may be instrumental in craniofacial and dental matrix maturation, potentially functioning in the maintenance of non-mineralized matrix.