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Modification of proteins by non-enzymatic glycation occurs in diabetes mellitus, aging and pathological states including renal failure. Glycation reactions span early reversible reactions to irreversibly modified species termed advanced glycation endproducts (AGEs). Long-lived extracellular matrix (ECM) proteins are particularly susceptible to AGE modification. As ECM proteins are known to regulate vascular function through binding with integrins, AGE formation may modify normal interactions between matrix and vascular smooth muscle cells (VSMCs) ultimately impacting vasoregulation. The purpose of this study was to selectively investigate the adhesion between either non-glycated or glycated fibronectin (FN) with VSMCs using Atomic Force Microscopy (AFM). AFM probes were functionalized with FN or glycated FN and cyclically interacted with isolated VSMCs using a force mode nano-indentation protocol. Adhesion was quantified as the rupture force required to disrupt adhesion bonds the formed during contact of the AFM probe with the cell and also as the probability of an adhesive interaction occurring. AFM probes coated with FN alone required 78.6 ± 19.9 pN (N=5) to disrupt adhesive bonds whereas glycated FN required 49.9 ± 5.7 pN (N=5). Binding probability was, however, significantly increased for AFM probes coated with glycated FN vs FN alone, 72.9 ± 3.5 % vs 63.0 ± 1.6 %, (N=5) respectively. Selectivity for binding to alpha5 integrin was investigated by adding a solubilized function-blocking antibody to the cell bath. Anti-alpha5 antibody significantly reduced binding to FN (14.27%) but had no effect on glycated FN. These results establish that glycation of ECM proteins can significantly affect their adhesive interaction with VSM and integrins and this could in turn lead to altered cell-integrin based regulation of VSM function. (Supp by P01 HL095486 and RO1 HL92241)