Regional gene therapy, which involves the delivery of growth factors to a specific anatomic site, has the potential to enhance bone formation in clinical application. Helper-dependent adenoviral vectors, which have deleted all of the viral coding regions, have been shown to be safe and highly efficient with long-lasting transgene expression. In this study, we constructed a helper-dependent adenoviral vector producing bone morphogenetic protein-2 (AdHDBMP-2). The AdHDBMP-2 increased the alkaline phosphatase activity of W-20-17 cells in vitro. In addition, when AdHDBMP-2 infected rat bone marrow cells were implanted into the hindlimbs of SCID mice, orthotopic bone formation was shown at 2 weeks. To our knowledge, this is the first study to demonstrate bone formation with the helper-dependent adenoviral vector with the BMP-2 expression cassette. This type of gene therapy vector could prove to be highly useful for bone augmentation in patients with bone loss associated with trauma, revision total joint arthroplasty, or cancer.