The interaction of fibroblasts with the extracellular matrix is critical for wound healing. Advanced glycation end products (AGEs) occur through nonenzymatic glycation of long-lived proteins such as collagens. One precursor to these modifications, 3-deoxyglucosone (3DG), is elevated in patients with diabetes and contributes to the accumulation of AGEs on collagen with increasing chronological age. Because wound repair is dependent on fibroblast migration, proliferation, and expression of extracellular matrix proteins, we examined the role of 3DG-treated collagen and the subsequent response of fibroblasts to this modification. We found that fibroblasts adhere more strongly to 3DG-treated collagen and do not migrate efficiently into the wound site. We further show that 3DG-treated collagen induces perinuclear localization of focal adhesion kinase and paxillin, indicative of decreased association of these proteins with the cytoskeleton. Additionally, these cells expressed higher levels of the misfolded indicator protein growth arrest and DNA damage inducible gene 153. These data suggest that fibroblast/matrix interactions are altered as AGEs accumulate and affect focal adhesion formation. Furthermore, 3DG may be a factor mediating chronic wounds observed in patients with diabetes and in the elderly by altering the signaling within the fibroblast and inducing the misfolding of proteins.