In the current study we examined whether diabetes affected the ability of HDL to stimulate nitric oxide production. Using HDL isolated from both diabetic humans and diabetic mouse models we found that female HDL no longer induced nitric oxide synthesis despite containing equivalent amounts of estrogen as non-diabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of nitric oxide generation. Analyses of both the human and mouse diabetic HDL particles demonstrated that the HDLs contained increased levels of myristic acid. To determine if myristic acid associated with HDL particles was responsible for the decrease in nitric oxide generation, myristic acid was added to HDL isolated from non-diabetic humans and mice. Myristic acid associated HDL inhibited the generation of nitric oxide in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial nitric oxide synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of nitric oxide production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with eNOS but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to demonstrate that a specific fatty acid associated with HDL inhibits the stimulation of nitric oxide generation. These findings have important implications regarding cardiovascular disease in diabetic patients.