Despite significant improvements, the current management of primary osteosarcoma is still limited by the development of metastatic disease, which occurs in approximately 30% of patients despite aggressive multiagent chemotherapy and tumor-ablative surgery. Therefore, there is a need for the development of novel agents to improve the outcome of these patients. Pigment epithelium-derived factor (PEDF) has been shown to be one of the most potent inhibitors of angiogenesis, and more recently has demonstrated a functional role in tumor growth, invasion and metastasis. In this study we report, for the first time, the multitargeted role of PEDF in the inhibition of growth, angiogenesis and metastasis of two orthotopic models of osteosarcoma (rat UMR 106-01 and human SaOS-2). Through stable plasmid-mediated gene transfer of full-length human PEDF, we show that PEDF overexpression significantly reduced tumor cell proliferation (P<0.05) and Matrigel invasion (UMR(PEDF), P<0.001; SaOS(PEDF), P<0.05) and increased adhesion to collagen type-1 (P<0.01), in vitro. In vivo, PEDF overexpression dramatically suppressed orthotopic osteosarcoma growth (P<0.05) and the development of spontaneous pulmonary metastases (UMR(PEDF), P<0.05; SaOS(PEDF), P<0.001). Furthermore, PEDF-overexpressing tumors exhibited reduced intratumoral angiogenesis, evidenced by a significant decrease in microvessel density (P<0.05). Therefore, together these results suggest that PEDF may be a new and promising approach for the treatment of osteosarcoma.