The VHL tumor suppressor gene is frequently inactivated in several human tumors, including bone sarcomas. We previously identified that reduced expression of VHL protein is implicated in sarcomagenesis. However, the underlying biological functions of restored VHL protein expression have not been clearly elucidated in bone sarcomas. Here we initially constructed a recombinant adenovirus 5-VHL vector (Ad5-VHL) and evaluated its expression in bone sarcomas, and antitumor activity in vitro and in vivo. We found that the adenovirus-mediated increase of VHL significantly suppresses bone sarcoma cell growth, attributed to induction of apoptosis mediated by increased caspase-3 activity and modulated Bcl-2 protein family. This suppression effect involves inhibition of Wnt/β-catenin signaling and upregulation of GSK-3β. Moreover, Ad5-VHL showed a dramatic antitumor effect on a chondrosarcoma xenograft model. These findings establish that Ad5-VHL suppresses bone sarcoma cell growth by inhibiting Wnt/β-catenin signaling, and may be a novel target for gene-based therapy of bone sarcomas.