AbstractBio–nano interfaces are integral to all applications of nanomaterials in biomedicine. In addition to peptide‐ligand‐functionalized nanomaterials, passivation on 2D nanomaterials has emerged as a new regulatory factor for integrin activation. However, the mechanisms underlying such ligand‐independent processes are poorly understood. Here, using graphene oxide passivated with polyethylene glycol (GO‐PEG) as a test bed, a ternary simulation model is constructed that also includes a membrane and both subunits of integrin αvβ8 to characterize GO‐PEG‐mediated integrin activation on the cell membrane in a ligand‐independent manner. Combined with the experimental findings, production simulations of the ternary model show a three‐phase mechanotransduction process in the vertical interaction mode. Specifically, GO‐PEG first induces lipid aggregation‐mediated integrin proximity, followed by transmembrane domain rotation and separation, leading to the extension and activation of extracellular domains. Thus, this study presents a complete picture of the interaction between passivated 2D nanomaterials and cell membranes to mediate integrin activation, and provides insights into the potential de novo design and rational use of novel desirable nanomaterials at diverse bio–nano interfaces.