Endothelial cell dysfunction is an early event in cardiovascular disease and atherosclerosis. The origin of this dysfunction is unresolved, but accumulating evidence implicates damaging oxidants, including hypochlorous acid (HOCl), a major oxidant produced by myeloperoxidase (MPO), during chronic inflammation. MPO is released extracellularly by activated leukocytes and binds to extracellular molecules including fibronectin, a major matrix glycoprotein involved in endothelial cell binding. We hypothesized that MPO binding might influence the modifications induced on fibronectin, when compared to reagent HOCl, with this including alterations to the extent of damage to protein side-chains, modified structural integrity, changes to functional domains, and impact on naïve human coronary artery endothelial cell (HCAEC) adhesion and metabolic activity. The effect of increasing concentrations of the alternative MPO substrate thiocyanate (SCN⁻), which might decrease HOCl formation were also examined. Exposure of fibronectin to MPO/H₂O₂/Cl⁻ is shown to result in damage to the functionally important cell-binding and heparin-binding fragments, gross structural changes to the protein, and altered HCAEC adhesion and activity. Differences were observed between stoichiometric, and above-stoichiometric MPO concentrations consistent with an effect of MPO binding to fibronectin. In contrast, MPO/H₂O₂/SCN⁻ induced much less marked changes and limited protein damage. Addition of increasing SCN⁻ concentrations to the MPO/H₂O₂/Cl⁻ system provided protection, with 20 μM of this anion rescuing damage to functionally-important domains, decreasing chemical modification, and maintaining normal HCAEC behavior. Modulating MPO binding to fibronectin, or enhancing SCN⁻ levels at sites of inflammation may therefore limit MPO-mediated damage, and be of therapeutic value.