Enhanced production of receptor activator of nuclear factor-kappaB ligand (RANKL) and its binding to RANK on the osteoclasts have been associated with osteolysis in breast cancer bone metastasis. Osteoprotegerin (OPG) is a decoy receptor that prevents RANKL-RANK interaction. This study determined the effects of sustained expression of OPG using a recombinant adeno-associated viral (rAAV) vector in mouse model of osteolytic breast cancer. Bone metastasis was established by intracardiac injection of the human breast cancer cell line MDA-MB-435. Following this, mice were administered a one-time intramuscular injection of rAAV encoding either OPG.Fc (OPG) or green fluorescent protein (GFP). Mice were killed 1 month later and the effects of therapy on tumor growth and bone remodeling were evaluated. Bioluminescence imaging showed significant reduction of tumor growth in bone of OPG.Fc-treated mice. Micro-computed tomography (microCT) analysis and histomorphometry of the tibia indicated significant protection of trabecular and cortical bones after OPG.Fc therapy. Despite the prevention of bone loss and tumor growth in bone, OPG.Fc therapy failed to provide long-term survival. OPG.Fc-treated mice developed more bone than age-matched normal mice, indicating a requirement for regulated transgene expression. Results of this study indicate the potential of rAAV-OPG therapy for reducing morbidity and mortality in breast cancer patients with osteolytic bone damage.