To elucidate a potential molecular link between diabetes and atherosclerosis, we investigated the role of Janus tyrosine kinase (JAK) for NAD(P)H oxidase-derived superoxide generation in the enhanced proliferative capacity of vascular smooth muscle cells (VSMC) of Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of type 2 diabetes. An enhanced proliferative response to 10% fetal bovine serum (FBS) and superoxide generation with an increased NAD(P)H oxidase activity were observed in diabetic (OLETF) VSMC. Both the enhanced proliferation and superoxide generation in diabetic VSMC were significantly attenuated by AG490, JAK2 inhibitor, and PP2, Src kinase inhibitor. Tyrosine phosphorylation of proteins in diabetic VSMC, especially JAK2, was increased compared to control VSMC. Furthermore, the enhanced NAD(P)H oxidase activity in diabetic VSMC was significantly attenuated by AG490 in a dose-dependent manner. Together, these results indicate that the signal pathway which leads to diabetes-associated activation of Src kinase/JAK is critically involved in the diabetic VSMC proliferation through NAD(P)H oxidase activation and superoxide generation.