Fibronectin coating increases implant biocompatibility by enhancing surface endothelialization via integrin-mediated binding. Surface properties determine the fibronectin orientation and conformation, dictating which ligands are presented, and therefore altering the bioactivity of an implant surface. In this study, polyurethane was treated with oxygen plasma, which allowed for a simultaneous modification of the surface chemistry and topography to modulate fibronectin adsorption. By varying the parameters of the treatment, human plasma fibronectin adsorbed on the surfaces in different conformations, orientations, and binding affinities, which was investigated by atomic force microscopy, fluorescence microscopy, monoclonal and polyclonal antibody staining and reflectometric interference spectroscopy. Apart from the most hydrophilic rough surfaces, the adsorbed fibronectin showed a lower binding affinity and less conformational change on the more hydrophilic surfaces. A large amount of exposed fibronectin-cell binding was detected on the rough treated and the smooth untreated surfaces. Primary isolated human umbilical vein and human microvascular endothelial cells showed a significantly higher cell adherence on the absorbed fibronectin with a low binding affinity and low conformational changes. Significant differences in the formation of mature focal adhesions and the reorganization of F-actin were identified on the rough treated and the smooth untreated surfaces. Our data suggest that oxygen plasma treatment is a reliable technique for the modulation of fibronectin adsorption in order to adjust fibronectin bioactivity and impact cell responses to implant surfaces.