Purpose Advanced glycation endproduct (AGE) formation on the basement membrane of retinal capillaries has been previously described but the impact of these adducts on capillary endothelial cell function vascular repair remains uncertain. This investigation has evaluated retinal microvascular endothelial cells (RMECs) growing on AGE-modified fibronectin (FN) and determined how this has an impact on cell-substrate interactions and downstream oxidative responses and cell survival. Methods RMECs were grown on methylglyoxal-modified FN (AGE-FN) or native FN as a control. RMEC attachment and spreading was quantified. In a separate treatment, the AGE-FN substrate had Arg-Gly-Asp-Ser (RGDS) or scrambled peptide added before seeding. Phosphorylation of focal adhesion kinase (FAK) and α5β1 integrin localization was assessed and apoptosis evaluated. In a subset of RMECs that remained attached to the AGE-FN substrate, the production of superoxide (O2-) was assayed using dihydroethidium (DHE) fluorescence or lucigenin, in the presence or absence of NADPH. The specificity of the O2- assays was confirmed by inhibition in the presence of polyethylene-glycol-superoxide dismutase (PEG-SOD). AGE-mediated changes to mRNAs encoding key basement membrane proteins and regulatory enzymes were investigated using real-time RT–PCR. Results AGE-FN reduced RMEC attachment and spreading when compared to FN controls (p<0.001). RGDS peptide enhanced cell attachment on AGE-FN (p<0.001), while the scrambled peptide had no effect. FAK phosphorylation in AGE-exposed RMECs was reduced in a time-dependent fashion, while α5β1 integrin-immunoreactivity became focal at the basal membrane. AGE-exposure induced apoptosis, a response significantly prevented by RGDS peptide. AGE-exposure caused a significant increase in basal O2- and NADPH-stimulated production by RMECs (p<0.01), while AGE-FN also increased basement membrane associated mRNA expression (p<0.05). Conclusions AGE substrate modifications impair the function of retinal capillary endothelium and their reparative potential in response to diabetes-related insults. Arginine-specific modifications alter vital endothelial cell interactions with the substrate. This phenomenon could play an important role in dysfunction and nonperfusion of retinal capillaries during diabetes.