Endothelial dysfunction (ED) is a crucial and initial stage for the development of cardiovascular diseases. Accumulated evidence has demonstrated causative links between cigarette smoke (CS) and ED. However, the underlying mechanisms remain largely unknown. Pyroptosis is a unique form of inflammatory cell death. In this study, we found that cigarette smoke extract (CSE) increased pyroptosis in endothelial cells (ECs) as evidenced by increasing lactate dehydrogenase release and the number of propidium iodide (PI) positive cells. A specific NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inhibitor (MCC950) pretreatment dramatically reduced CSE-induced pyroptosis. Additionally, we also observed that N-Acetylcysteine (NAC, a ROS scavenger) pretreatment inhibited NLRP3 inflammasome activation as evidenced by suppressing the upregulation of NLRP3, ASC, cleaved-caspase-1, GSDMD-N, IL-1β and IL-18 protein levels in CSE-treated ECs. Meanwhile, NAC pretreatment also remarkably inhibited CSE-induced EC pyroptosis. Melatonin is a hormone synthesized and secreted by mammalian pineal gland and plays a protective role in various cardiovascular diseases through its powerful anti-inflammatory and antioxidant properties. In this study, melatonin was observed to inhibit ROS production, NLRP3 inflammasome activation and pyroptosis in CSE-treated ECs. Moreover, oxidative stress and NLRP3 inflammasome activation in carotid arteries of smoking rats was also inhibited by melatonin. In conclusion, our study generated two novel findings, (i) CS activates ROS/NLRP3 axis and induces EC pyroptosis; (ii) melatonin attenuates CS-induced EC pyroptosis by inhibiting ROS/NLRP3 axis.