||Cardiovascular complications are the leading cause of death in individuals with conditions associated with insulin resistance, e.g. type 2 diabetes, diet-induced obesity. In patients with type 2 diabetes, cardiovascular complications are 2-4 times more prevalent. One common cardiovascular complication in patients with type 2 diabetes is arterial dysfunction. It has been shown that free fatty acids (FFAs) are elevated in patients with type 2 diabetes and these individuals exhibit arterial dysfunction. The activity of the endothelial nitric oxide (NO) synthase (eNOS) enzyme is impaired in arteries from these patients. Earlier we showed that a metabolite of FFAs - ceramide - contributes to arterial dysfunction in rodents with diet-induced obesity and type 2 diabetes, by impairing eNOS enzyme function. The aims of the present study were two-fold. First, we sought to determine how ceramide impairs eNOS enzyme function. Second, after revealing the involvement of protein phosphatase 2A (PP2A) in this process, we sought to determine how ceramide activates PP2A. eNOS is responsible for NO production, which is essential for promoting vasodilation and opposing vasoconstriction. eNOS is regulated, at least in part, by kinases and phosphatases. Protein phosphatase 2A (PP2A) is the most abundant phosphatase in the cytosol and it is responsible for dephosphorylating eNOS directly and other kinases and proteins that are necessary for full eNOS enzyme function. Our first hypothesis was that ceramide elevates PP2A activity, which subsequently impairs eNOS enzyme function by binding directly to the enzyme complex. We observed that elevated ceramide causes PP2A to associate directly with eNOS, which subsequently disrupts important protein-protein interactions within the eNOS enzyme complex to ultimately lower NO production. Under physiological conditions, PP2A is bound/restrained by inhibitor 2 of PP2A (I2PP2A) in the cytosol. We next tested the hypothesis that ceramide activates PP2A by disrupting the interaction between I2PP2A and PP2A. We observed that when ceramide is elevated, the inhibitory association between I2PP2A and PP2A is disrupted. Taken together, elevated ceramide removes the restraint of I2PP2A on PP2A, which facilitates the binding of PP2A with eNOS to an extent that disrupts eNOS enzyme function. By determining these responses, we have provided insight into a new therapeutic target - PP2A - that can be examined more thoroughly for the treatment of vascular disease in the context of type 2 diabetes.