Endothelial dysfunction is a typical characteristic of sepsis. Endothelial nitric oxide synthase (eNOS) is important for maintaining endothelial function. Our previous study reported that the NLRP3 inflammasome promoted endothelial dysfunction by enhancing inflammation. However, the effects of NLRP3 on eNOS require further investigation. Therefore, the present study aimed to investigate the role of NLRP3 on eNOS expression levels in cecal ligation and puncture-induced impaired endothelium-dependent vascular relaxation and to determine the protective effects of melatonin. eNOS expression levels were discovered to be downregulated in the mesenteric arteries of sepsis model mice. Inhibiting NLRP3 with 10 mg/ kg MCC950 or inhibiting IL-1β with 100 mg diacerein rescued the eNOS expression and improved endothelium-dependent vascular relaxation. In vitro, IL-1β stimulation downregulated eNOS expression levels in human aortic endothelial cells (HAECs) in a concentration- and time-dependent manner, while pretreatment with 1 µM of the proteasome inhibitor MG132 reversed this effect. In addition, treatment with 10 mg/kg MG132 also prevented the proteolysis of eNOS and improved endothelium-dependent vascular relaxation in vivo. Notably, treatment with 30 mg/kg melatonin downregulated NLRP3 expression levels and decreased IL-1β secretion, subsequently increasing the expression of eNOS and improving endothelium-dependent vascular relaxation. In conclusion, the findings of the present study indicated that the NLRP3/IL-1β axis may impair vasodilation by promoting the proteolysis of eNOS and melatonin may protect against sepsis-induced endothelial relaxation dysfunction by inhibiting the NLRP3/IL-1β axis, suggesting its pharmacological potential in sepsis.