The ability of bone to resist catastrophic failure is critically dependent upon the material properties of bone matrix, a composite of hydroxyapatite, collagen type I, and noncollagenous proteins. These properties include elastic modulus, hardness, and fracture toughness. Like other aspects of bone quality, matrix material properties are biologically-defined and can be disrupted in skeletal disease. While mineral and collagen have been investigated in greater detail, the contribution of noncollagenous proteins such as osteopontin to bone matrix material properties remains unclear. Several roles have been ascribed to osteopontin in bone, many of which have the potential to impact material properties. To elucidate the role of osteopontin in bone quality, we evaluated the structure, composition, and material properties of bone from osteopontin-deficient mice and wild-type littermates at several length scales. Most importantly, the results show that osteopontin deficiency causes a 30% decrease in fracture toughness, suggesting an important role for OPN in preventing crack propagation. This significant decline in fracture toughness is independent of changes in whole bone mass, structure, or matrix porosity. Using nanoindentation and quantitative backscattered electron imaging to evaluate osteopontin-deficient bone matrix at the micrometer level, we observed a significant reduction in elastic modulus and increased variability in calcium concentration. Matrix heterogeneity was also apparent at the ultrastructural level. In conclusion, we find that osteopontin is essential for the fracture toughness of bone, and reduced toughness in osteopontin-deficient bone may be related to the increased matrix heterogeneity observed at the micro-scale. By exploring the effects of osteopontin deficiency on bone matrix material properties, composition and organization, this study suggests that reduced fracture toughness is one mechanism by which loss of noncollagenous proteins contribute to bone fragility.