IGF-I and -II provide paracrine and autocrine stimuli, respectively, for extravillous trophoblast (EVT) cell migration. This study examined the role of α_vβ₃ integrin and its signaling pathway in IGF-I-stimulated migration. Migration assays were conducted using cultured EVT cells treated with or without IGF-I in the presence or absence of αIR3, Arg-Gly-Asp (RGD) hexapeptide, and antibody against α_vβ₃ integrin. Morphological changes were studied using scanning electron microscopy. Colocalization of α₅β₁ α_vβ₃ integrins, vinculin, focal adhesion kinase, and paxillin were determined by immuno-cytochemistry and immunoblotting. The results showed that IGF-I could stimulate EVT cell migration in a time- and dose-dependent manner and addition of αIR3, Arg-Gly-Asp hexapeptide, and antibody against α_vβ₃ integrin attenuated the IGF-I migratory effect. Scanning electron microscopy images revealed that IGF-I promoted lamellipodia formation. Immunostaining and immunoblotting exhibited the colocalization of α_vβ₃ integrin with phosphorylated focal adhesion kinase, paxillin, and vinculin at focal adhesions after IGF-I treatment. Immunoblotting demonstrated an increase in focal adhesion kinase and paxillin tyrosine phosphorylation followed by tyrosine phosphorylation of IGF-I receptor in a time- and dose-dependent manner. These findings indicated α_vβ₃ integrin localization in the core of focal adhesions of EVT cells and that α_vβ₃ integrin signaling pathways are activated in IGF-I-mediated migration of these cells.