Biomineralization is a complex process in the development of mineralized tissues such as bone and pathological calcifications such as atherosclerotic plaques, kidney stones and gout. Osteopontin (OPN), an anionic phosphoprotein, is expressed in mineralizing tissues and has previously been demonstrated to be a potent inhibitor of hydroxyapatite formation. The OPN-deficient (Opn−/−) mouse displays a hypermineralized bone phenotype starting at 12 weeks postnatally. By isolating and culturing Opn−/− and wild-type (WT) osteoblasts, we sought to determine the role of OPN and two of its functional peptides in osteoblast development and mineralization. Opn−/− osteoblasts had significantly increased mineral deposition relative to their WT counterparts, with no physiologically relevant change in gene expression of osteogenic markers. Supplementation with bovine milk OPN (mOPN) led to a dramatic reduction in mineral deposition by the Opn−/− osteoblasts. Treatment with OPN-derived peptides corresponding to phosphorylated OPN-(220–235) (P3) and non-phosphorylated OPN-(65–80) (OPAR) also rescued the hypermineralization phenotype of Opn−/− osteogenic cultures. Supplementation with mOPN or the OPN-derived peptides did not alter the expression of terminal osteogenic markers. These data suggest that OPN plays an important role in the regulation of biomineralization, but that OPN does not appear to affect osteoblast cell development in vitro.