Osteosarcoma is the most frequent primary bone tumor that develops mainly during youth, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% after 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options. The anti-bone resorption molecule receptor activator of NF-kappaB (RANK) is very promising, as it may block the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. The cDNA encoding murine RANK-Fc (mRANK-Fc) was administered by gene transfer using an amphiphilic polymer in a mouse model of osteolytic osteosarcoma. Clinical and bone microarchitecture variables were assessed by radiography and micro-CT analyses. In vitro experiments were designed to determine the mechanism of action of RANK-Fc on tumor cell proliferation (XTT assays), apoptosis (caspase activation), cell cycle distribution (fluorescence-activated cell sorting analysis), or gene expression (reverse transcription-PCR). RANK-Fc was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development and in reducing the tumor incidence, the local tumor growth, and the lung metastases dissemination leading to a 3.9-fold augmentation of mice survival 28 days after implantation. On the contrary, mRANK-Fc did not prevent the development of nonosseous tumor nodules, suggesting that bone environment is necessary for mRANK-Fc therapeutic efficacy. Furthermore, mRANK-Fc has no direct activity on osteosarcoma cells in vitro. mRANK-Fc exerts an indirect inhibitory effect on osteosarcoma progression through inhibition of bone resorption. [Mol Cancer Ther 2008;7(10):3389-98].