Special AT-rich sequence-binding protein 2 (SATB2 ) acts as a potent transcription factor to promote osteoblast differentiation and bone regeneration. In this study, we first used lentiviral-mediated gene transfer of Satb2 into mouse bone marrow stromal cells (BMSCs) and investigated the capacity of SATB2 overexpression to promote osteogenic differentiation in vitro and in vivo. We found that LV-Satb2 -transduced BMSCs produced SATB2 protein and underwent rapid and marked osteogenic differentiation, as demonstrated by increased expression of osteoblastic genes, including runt-related transcription factor 2 (Runx2), transcription factor Sp7 (Sp7), activating transcription factor 4 (Atf4), and bone sialoprotein (Bsp), and increased alkaline phosphatase activity and Alizarin Red S staining. To analyze the induction of bone formation in vivo, LV-Satb2-transduced BMSCs were implanted into the hindlimbs of syngeneic mice, with β-tricalcium phosphate as the scaffolding material. Four weeks after implantation, transduction with LV-Satb2 had greatly enhanced the formation of new bone. These data demonstrated the capacity of lentiviral-mediated SATB2 to promote the osteogenic differentiation of BMSCs in vitro and to enhance bone formation through a tissue-engineering technique that may be useful in bone-regenerative medicine.