Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PPi), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PPi, resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl−/− mice, a model for infantile HPP. In Alpl−/− mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl−/− mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl−/− molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl−/− dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl−/− mice, was present in the generally unmineralized Alpl−/− mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl−/− mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl−/− mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PPi during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients. © 2013 American Society for Bone and Mineral Research