Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding­ and fibrillogenesis through binding to α5β1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif–deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin–binding sites. Our results demonstrate that α5β1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5β1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins.