Purpose: The clinical course of osteosarcoma (OS) demands the development of new therapeutic options. Conditionally replicative adenoviruses (CRAds) represent promising agents for the treatment of solid tumors, because CRAds have an intrinsic replication capacity that allows in situ amplification and extensive tumor infection through lateral spread. The CRAd AdΔ24 has been developed to replicate selectively in cells with a defective retinoblastoma (Rb) pathway. Because genetic alterations in the Rb pathway are frequently observed in OS, AdΔ24 might be useful in the treatment of this cancer. Experimental Design: Because the lack of Coxsackie adenovirus receptor on OS cells limits the efficacy of CRAd treatment, we explored alternative target molecules on OS. Insertion of an Arg-Gly-Asp (RGD-4C) integrin-targeting motif into the adenovirus fiber knob expanded tropism toward the α vβ3 and αvβ5 integrins. The oncolytic capacity of the CRAd Ad5-Δ24RGD was tested on primary OS cells in vitro and in vivo. Results: The αvβ 3 and αvβ5 integrins are abundantly expressed on OS cells. RGD-mediated infection augmented adenovirus infection of primary OS cells by two orders of magnitude. Ad5-Δ24RGD was shown to be highly active in killing human OS cell lines, as well as primary cell cultures. Furthermore, intratumoral injections with Ad5-Δ24RGD into established human OS xenografts that were derived directly from a patient with OS refractory for chemotherapeutic treatment caused a significant tumor-growth delay. Furthermore, adenoviral particles could be detected in tumor cells 25 days posttumor injection. Conclusions: Targeting adenovirus toward integrins rendered OS cells more sensitive to killing by Ad5-Δ24RGD. These findings suggest that Ad5-Δ24RGD has potential for use in OS treatment.