UNLABELLED: OPN is an ECM protein with diverse localization and functionality. The role of OPN during fracture healing was examined using wildtype and OPN(-/-) mice. Results showed that OPN plays an important role in regulation of angiogenesis, callus formation, and mechanical strength in early stages of healing and facilitates late stage bone remodeling and ECM organization. INTRODUCTION: Osteopontin (OPN) is an extracellular matrix (ECM) protein with diverse localization and functionality that has been reported to play a regulatory role in both angiogenesis and osteoclastic bone remodeling, two vital processes for normal bone healing. MATERIALS AND METHODS: Bone repair in wildtype and OPN(-/-) mice was studied using a femoral fracture model. microCT was used for quantitative angiographic measurements at 7 and 14 days and to assess callus size and mineralization at 7, 14, 28, and 56 days. Biomechanical testing was performed on intact bones and on fracture specimens at 14, 28, and 56 days. Histology and quantitative RT-PCR were used to evaluate cellular functions related to ECM formation and bone remodeling. RESULTS: OPN deficiency was validated in the OPN(-/-) mice, which generally displayed normal levels of related ECM proteins. Intact OPN(-/-) bones displayed increased elastic modulus but decreased strength and ductility. Fracture neovascularization was reduced at 7 but not 14 days in OPN(-/-) mice. OPN(-/-) mice exhibited smaller fracture calluses at 7 and 14 days, as well as lower maximum torque and work to failure. At 28 days, OPN(-/-) mice had normal callus size but a persistent reduction in maximum torque and work to failure. Osteoclast differentiation occurred normally, but mature osteoclasts displayed reduced functionality, decreasing late stage remodeling in OPN(-/-) mice. Thus, at 56 days, OPN(-/-) fractures possessed increased callus volume, increased mechanical stiffness, and altered collagen fiber organization. CONCLUSIONS: This study showed multiple, stage-dependent roles of OPN during fracture healing. We conclude that OPN deficiency alters the functionality of multiple cell types, resulting in delayed early vascularization, altered matrix organization and late remodeling, and reduced biomechanical properties. These findings contribute to an improved understanding of the role of OPN in vivo and provide new insight into mechanistic control of vascularization and bone regeneration during fracture repair.