Insulin-like growth factor binding protein-3 (IGFBP-3), a major IGF-binding protein in human serum, regulates the growth of non-small cell lung cancer (NSCLC) cells through IGF-dependent and IGF-independent mechanisms. However, the role of IGFBP-3 in lung cancer metastasis is not well known. In the present study, we showed that noncytotoxic doses of adenoviral or recombinant IGFBP-3 significantly decreased the migration and invasion of H1299 and A549 NSCLC cells. Furthermore, treatment of human lung fibroblasts with recombinant IGFBP-3 suppressed their ability to stimulate the invasion of H1299 cells. Overexpression of IGFBP-3 markedly reduced lung metastasis of A549 cells in an experimental animal model system and prolonged the survival time of the animals. Urokinase-type plasminogen activator (uPA) inhibitor treatment or uPA small interfering RNA transfection of A549 and H1299 cells resulted in a significant decrease in invasion. Corresponding ELISA, Western blot, gelatin zymogram, and semiquantitative reverse transcription-PCR analyses revealed that IGFBP-3 reduced the expression of uPA mRNA through IGF-independent mechanisms. The specific role of uPA in anti-invasive activity of IGFBP-3 was further confirmed in NSCLC cells, in which uPA expression/activity was suppressed by the transfection with synthetic small interfering RNA or by the treatment with uPA inhibitor or induced by the infection with an adenoviral vector. IGFBP-3 also decreased the expression/activity of matrix metalloproteinase-2 through IGF-dependent but uPA-independent pathways. Taken together, our data suggest that IGFPB-3 effectively block uPA- and matrix metalloproteinase-2-stimulated invasion pathways, ultimately reducing lung cancer cell metastasis. Our findings indicate that IGFBP-3 may be a promising anti-invasive and antimetastatic therapeutic agent in lung cancer.