Severe dental troubles are associated with X-linked hypophosphatemic rickets and are mainly related to impaired dentin mineralization. In dentin matrix, matrix extracellular phosphoglycoprotein (MEPE) may be protected from proteolysis by a specific interaction with PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome). The objective of our work was to determine whether PHEX impairment induces MEPE cleavage in dentin and the subsequent release of the C-terminal acidic serine- and aspartate-rich motif (ASARM) peptide, which is known to inhibit mineralization. By Western blot analysis, we explored dentin extracts from seven hypophosphatemic patients with mutations of the PHEX gene. A proteomic approach combining immunoprecipitation, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry and matrix-assisted laser desorption ionization-time of flight analysis of the samples completed this exploration. This study shows a 4.1-kDa peptide containing the MEPE-derived ASARM peptide in hypophosphatemic samples. The presence of ASARM was less marked in patients treated with 1-hydroxylated vitamin D and phosphate during growth. Moreover, recombinant ASARM implanted in a rat pulp injury model disturbed the formation of the reparative dentin bridge. These results suggest that abnormal MEPE cleavage occurs when PHEX activity is deficient in humans, the ASARM peptide may be involved in the mineralization defects and the PHEX-MEPE interaction may be indirect, as ensuring a better phosphate and vitamin D environment to the mineralizing dentin prevents MEPE cleavage.