Cytoskeletal restraints affect force-regulated integrin function in cell adhesion. However, the structural and molecular basis underlying the effect of cytoskeletal restraints on beta1 integrin binding to fibronectin is still largely unknown. In this study, we used steered molecular dynamics simulations to investigate the changes in glycosylated beta1 integrin-fibronectin binding and in conformation and structure of the glycosylated beta1 I-like domain-FN-III9-10 complex caused by altered restraints applied to beta1 I-like domain. The results revealed that imposition of the increased constraints on beta1 integrin increased resistance to force-induced dissociation of the beta1 I-like domain-fibronectin complex. Specifically, the increased constraints enhanced resistance to relative conformational changes in the RGD-synergy site in fibronectin, increased the conformational stability of fibronectin, and prevented losses in hydrogen bond occupancy of each beta-strand pair in FN-III10 resulting from external force. The increased constraints also resulted in an increase in correlated motion between residues in the beta1 I-like domain, which may directly affect the interaction of beta1 integrin with fibronectin. Results from this study provide molecular and structural insights into the effects of altered restraints in beta1 integrin on the interaction between glycosylated beta1 Integrin and fibronectin and its induced cell adhesion.