Endothelial dysfunction induced by chronic inflammation has been considered one of the most important mechanisms behind a variety of cardiovascular diseases. Extensive efforts have been made in past decades to explore the underlying mechanisms of endothelial dysfunction and to develop new therapeutic agents for the treatment of cardiovascular diseases. Zafirlukast, a selective antagonist of CysLT receptor 1 (CysLT1R), has been licensed by the U.S. Food and Drug Administration (FDA) for the treatment of asthma. In this study, we found that zafirlukast possesses beneficial protective effects on endothelial cells from TNF-α-induced inflammatory response and injury. Our results indicate that TNF-α treatment induces CysLT1R expression. The addition of zafirlukast to culture media suppressed TNF-α-induced expression of endothelial vascular adhesion molecules, such as ICAM-1, VCAM-1, and induction of cytokines, including IL-1β, IL-6, and IL-8. Zafirlukast also ameliorated production of reactive oxygen species (ROS) and adhesion of monocytes to endothelial cells induced by TNF-α. Mechanistically, we demonstrate that zafirlukast suppresses MAPK kinase p38 and NF-κB activation to inhibit inflammatory mediators. Collectively, our findings provide insights into the mechanisms of a potential therapeutic strategy for endothelial dysfunction-related diseases and shed light on the possible application of zafirlukast in cardiovascular diseases such as atherosclerosis.