© 2017 The Protein Society Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn2+-bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf-2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion-dependent adhesion site, stabilized Mn2+ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the “outside-in” activation mechanism of integrin αVβ3 by binding to fibronectin.