Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding to fibronectin (Fn). Recognition of both a RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9) provide selectivity of α5β1 for fibronectin. Here, we have compared synergy site mutations and Fn truncation mutations for their α5β1 binding affinities and stabilities and interrogated Fn and α5β1 binding using hydrogen deuterium exchange mass spectrometry (HDX-MS) to gain insights into binding sites and the effects of ligand binding on integrin conformational change. Our results suggest that the synergistic effect of Fn9 requires both specific residues and a folded domain and that some residues that were considered to contribute to synergy are required for stability. HDX changes in fibronectin are localized to a peptide containing contacting residues in two β-strands and an intervening loop in Fn9 and the RGD containing loop in Fn10, indicative of binding sites. Decreases in HDX in α5β1 were found in an α5 peptide that associates with the Fn9 domain and inferred from a decrease in β1 subunit α1 helix exchange relative to an RGD peptide ligand. However, the dominant effect of Fn binding was an increase in deuterium exchange in α5β1 that was distributed over six sites in β1 and one in α5 that undergo changes in conformation or solvent accessibility and one unexpected site located in a contact between the α5 thigh and β-propeller domains.