Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p<0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p<0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, β1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-β but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.