Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice, osteoblast differentiation is impaired, and bone formation is absent. We hypothesized that overexpression of Osx in bone marrow-derived mesenchymal stem cells (BMSCs) would enhance osteogenic differentiation during bone regeneration in vivo. Overexpression of Osx in mouse BMSCs was achieved using retroviral infection together with a green fluorescent protein (GFP) vector to monitor transduction efficiency and determine the source of regenerative cells in implantation studies. Bone regeneration in vivo was evaluated by implanting BMSCs overexpressing Osx into 4-mm calvarial bone defects in adult mice using type I collagen sponge as a carrier. New bone formation in the defects was quantified using radiological and histological procedures 5 weeks after implantation. The results showed that implantation of Osx-transduced BMSCs resulted in 85% healing of calvarial bone defects as detected using radiological analyses. Histological examination of the implants demonstrated that the Osx-transduced group exhibited amounts of newly formed bone that was five times as high as in a group transduced with the empty vector. Immunohistochemistry for GFP showed positive immunoreaction localized to areas of newly engineered bone in the Osx-transduced group. Immunohistochemistry with antibodies against the extracellular matrix protein bone sialoprotein resulted in strong staining in areas of new bone formation. In addition, the clonal BMSCs showed an osteogenic potential similar to that of primary cultures of BMSCs, suggesting the usefulness of this model in bone tissue engineering. These results indicate that ex vivo gene therapy of Osx is a useful therapeutic approach in regenerating adult bone tissue.