Bone marrow stromal cells (BMSCs) are expected to be a source for tissue regeneration because they can differentiate into multiple cell types. Establishment of efficient gene transfer systems for BMSCs is essential for their application to regenerative medicine. In this study, we compared the transduction efficiency in mouse primary BMSCs by using fiber-modified adenovirus (Ad) vectors, and demonstrated that AdK7, which harbors a polylysin (K7) peptide in the C-terminus of the fiber knob, could efficiently express a transgene in BMSCs. Notably, AdK7 robustly drove transgene expression in more than 90% of the BMSCs at 3,000 vector particles/cell. Furthermore, we showed that in vitro and in vivo osteogenic potential of BMSCs was dramatically promoted by the transduction of Runx2 gene using AdK7. These results indicate that this transduction system could be a powerful tool for therapeutic applications based on BMSCs.