Hyperglycemia-induced oxidative stress and inflammation are central in the genesis of diabetic vascular complications. Toll-like receptors (TLRs) play a crucial role in promoting inflammatory responses and are known to be activated in diabetic patients. Also in animal models, they have been shown to have a role in the pathogenesis of diabetic vasculopathies. However, the mechanisms underlying this increase in TLR activity in diabetes are not well documented. Since increased reactive oxygen species (ROS) are also produced in various tissues under diabetic conditions, we postulated that ROS act as a potential activator of TLR. Several studies support our hypothesis that hyperglycemia-induced oxidative stress appears to be an important factor in promoting TLR activity in monocytes, both microvascular and macrovascular endothelial cells and cardiomyocytes and in animal models. Most importantly, the increase in ROS and TLR activity is ameliorated with antioxidant strategies. Thus, targeting ROS/NADPH oxidase with small molecular inhibitors could be a promising strategy to reduce both oxidative stress and TLR-mediated inflammation in diabetic vascular diseases.