Accumulating clinical evidence indicates that both type II diabetic individuals and glucose-intolerant individuals have increased aortic stiffness, an independent risk factor for cardiovascular and all-cause mortality. The present study sought to determine whether increased mechanical stiffness, an altered extracellular matrix, and a pro-fibrotic gene expression profile could be observed in the aorta of the insulin resistant Zucker fa/fa rat. Mechanical testing of Zucker fa/fa aortas showed increased vascular stiffness in both longitudinal and circumferential directions as compared to Zucker Lean controls. Unequal elevations in developed strain favoring the longitudinal direction resulted in a loss of anisotropy. Real-time quantitative PCR and immunohistochemistry revealed increased expression of fibronectin and collagen IV3 in the Zucker fa/fa aorta. In addition, expression of transforming growth factor beta-1(TGF1) and several Smad proteins was increased in vessels from insulin resistant animals. Exposure of rat vascular smooth muscle cells to 12-18 hours of insulin (100nmol/L) enhanced TGF1 mRNA expression, implicating a role for hyperinsulinemia in vascular stiffness. Thus, there is mechanical, structural, and molecular evidence of arteriosclerosis in the Zucker fa/fa rat at the glucose-intolerant, hyperinsulinemic stage.