Endothelial dysfunction precedes atherosclerosis and may constitute a critical link between obesity-related inflammation and cardiovascular disease. Neutrophil extracellular traps (NETs) have been shown to promote vascular damage in murine models of autoimmune disease and atherosclerosis. The impact of NETs towards endothelial dysfunction associated with obesity is unknown. Using a diet-induced obesity (DIO) mouse model, this study investigated whether the inhibition or degradation of NETs could reduce the endothelial dysfunction observed in DIO mice. Following induction of DIO, there were elevated plasma concentrations of monocyte chemoattractant protein-1 (MCP-1) and impairment of mesenteric arteriolar vasorelaxation in response to acetylcholine as measured by pressure myography. A marker of NET formation, cathelicidin-related antimicrobial peptide (CRAMP), was markedly increased in mesenteric arterial walls of DIO mice compared to mice on standard chow. Prevention of NET formation with Cl-amidine or dissolution of NETs with DNase restored endothelium-dependent vasodilation to the mesenteric arteries of DIO mice. These findings suggest an instrumental role for NETs in obesity-induced endothelial dysfunction.