The SIBLING family (small integrin‐binding ligand N‐linked glycoproteins) of mineral‐regulating proteins, which includes matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), contains an acidic serine‐ and aspartate‐rich motif (ASARM). X‐linked hypophosphatemia caused by inactivating mutations of the PHEX gene results in elevated mineralization‐inhibiting MEPE‐derived ASARM peptides. Although the OPN ASARM motif shares 60% homology with MEPE ASARM, it is still unknown whether OPN ASARM similarly inhibits mineralization. In this study we have examined the role of OPN ASARM and its interaction with PHEX enzyme using an osteoblast cell culture model, mass spectrometry, mineral‐binding assays, and computational modeling. MC3T3‐E1 osteoblast cultures were treated with differently phosphorylated OPN ASARM peptides [with 5 phosphoserines (OpnAs5) or 3 phosphoserines (OpnAs3)] or with control nonphosphorylated peptide (OpnAs0). Phosphorylated peptides dose‐dependently inhibited mineralization, and binding of phosphorylated peptides to mineral was confirmed by a hydroxyapatite‐binding assay. OpnAs0 showed no binding to hydroxyapatite and did not inhibit culture mineralization. Computational modeling of peptide‐mineral interactions indicated a favorable change in binding energy with increasing phosphorylation consistent with hydroxyapatite‐binding experiments and inhibition of culture mineralization. Addition of PHEX rescued inhibition of mineralization by OpnAs3. Mass spectrometry of cleaved peptides after ASARM‐PHEX incubations identified OpnAs3 as a PHEX substrate. We conclude that OPN ASARM inhibits mineralization by binding to hydroxyapatite in a phosphorylation‐dependent manner and that this inhibitor can be cleaved by PHEX, thus providing a mechanistic explanation for how loss of PHEX activity in X‐linked hyposphosphatemia can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia. © 2010 American Society for Bone and Mineral Research