Communication between cells and the extracellular matrix (ECM) is critical for regulation of cell growth, survival, migration, and differentiation. Remodeling of the ECM can occur under normal physiological conditions, as a result of tissue injury, and in certain pathological conditions. ECM remodeling leads to alterations in ECM composition and organization that can alter many aspects of cell behavior, including cell migration. The cell migratory response varies depending upon the type, the amount and organization of ECM molecules present, as well as the integrin and proteoglycan repertoire of the cells. We and others have shown that the deposition of several ECM molecules, including types I and III collagen, depends upon the presence and stability of ECM fibronectin. Hence, the effect of fibronectin and fibronectin matrix on cell function may partially depend upon its ability to direct the deposition of collagen in the ECM. In this manuscript, we use collagen-binding fibronectin mutants and recombinant peptides that interfere with fibronectin-collagen binding to show that fibronectin-dependent collagen I deposition regulates the cell migratory response to fibronectin. These data show that the ability of fibronectin to organize other proteins in the ECM is an important aspect of fibronectin function, and highlight the importance of understanding how interactions between ECM proteins influence cell behavior.