Homocystinuria is a genetic disorder resulting in elevated levels of homocysteine in plasma and tissues. Some of the skeletal and ocular symptoms such as long bone overgrowth, scoliosis, and ectopia lentis overlap with symptoms seen in Marfan syndrome. Marfan syndrome is caused by mutations in the extracellular matrix protein fibrillin-1. We previously showed that fibrillin-1 is a target for homocysteine and that the deposition of homocysteinylated fibrillin-1 in the extracellular matrix is compromised. Since the assembly of fibrillin-1 is critically dependent on fibronectin, we analyzed the consequences of fibronectin homocysteinylation and its interaction with fibrillin-1. Cellular fibronectin and proteolytic fragments were homocysteinylated and tested in various interaction assays with recombinant fibrillin-1 and heparin. Fibronectin homocysteinylation consistently compromised the fibronectin-fibrillin-1 interaction, while the interaction with heparin was not affected. Fibronectin homocysteinylation, but not cysteinylation, reduced the fibronectin dimers to monomers as shown by Western blotting. ELISA analyses of homocysteinylated fibronectin with three monoclonal antibodies demonstrated structural changes in the disulfide-containing FNI domains FNI(2), FNI(4), and FNI(9). Using fluorescently labeled fibronectin, we studied the consequence of fibronectin homocysteinylation on assembly in cell culture. Modified fibronectin showed deficiencies in denovo matrix incorporation and initial assembly. In conclusion, we define here characteristic structural changes of fibronectin upon homocysteinylation that translate into functional deficiencies in the fibronectin-fibrillin-1 interaction and in fibronectin assembly. Since fibronectin is a major organizer of various extracellular protein networks, these structural and functional alterations may contribute to the pathogenesis of homocystinuria and Marfan syndrome.