We have investigated whether systemic delivery of an oncolytic adenovirus, Ad.sTβRFc, expressing the soluble form of transforming growth factor-β receptor II fused with human immunoglobulin Fc fragment (sTGFβRIIFc), could inhibit breast cancer bone metastasis in a mouse model. MDA-MB-231 (human breast cancer) cells were inoculated into the left heart ventricles of nude mice. Once the skeletal tumors were visible by X-rays, mice were intravenously injected with either buffer, Ad.sTβRFc, Ad(E1⁻).sTβRFc (a replication-deficient adenovirus expressing sTGFβRIIFc), or Ad.luc2 (a replicating adenovirus expressing firefly luciferase gene). On days 2 and 7 after viral injections, viral replication and sTGFβRIIFc expression were detected in the skeletal tumors in Ad.sTβRFc-treated group; only viral replication in Ad.luc2 group, and sTGFβRIIFc expression in the Ad(E1⁻).sTβRFc group, were detected. To examine the therapeutic effects, buffer or various viral vectors were administered on days 4 and 7 after intracardiac injection of MDA-MB-231 cells. On day 28, X-ray radiography showed a highly significant reduction in lesion size by Ad.sTβRFc, a significant reduction by Ad.luc2, and some reduction by Ad(E1⁻).sTβRFc. Goldner's trichrome and hematoxylin-eosin staining of the bone sections revealed a significant reduction of tumor burden in the Ad.sTβRFc group, but not in the Ad(E1⁻).sTβRFc or Ad.luc2 group. There were significant reductions in free calcium levels by Ad.sTβRFc, Ad(E1⁻).sTβRFc, and Ad.luc2; however, only in the Ad.sTβRFc group were calcium levels reduced to the normal values. These results suggest that concomitant viral replication and sTGFβRIIFc production are important to inhibit bone metastasis and osteolysis, and that Ad.sTβRFc could be developed for targeting breast cancer bone metastases.