The Nod-like receptor family PYRIN domain containing 3 (NLRP3) inflammasome is a multiprotein signaling complex that plays a pivotal role in innate immunity, and the dysregulated NLRP3 inflammasome activation is implicated in various diseases. Tiliroside is a natural flavonoid in multiple medicinal and dietary plants with known anti-inflammatory activities. However, its role in regulating NLRP3 inflammasome activation and NLRP3-related disease has not been evaluated. Herein, it was demonstrated that tiliroside is inhibitory in activating the NLRP3 inflammasome in macrophages. Mechanistically, tiliroside promotes AMP-activated protein kinase (AMPK) activation, thereby leading to ameliorated mitochondrial damage as evidenced by the reduction of mitochondrial reactive oxygen species (ROS) production and the improvement of mitochondrial membrane potential, which is accompanied by attenuated NLRP3 inflammasome activation in macrophages. Notably, tiliroside potently attenuated lipopolysaccharide (LPS)-induced acute lung injury in mice, which has been known to be NLRP3 inflammasome dependent. For the first time, this study identified that tiliroside is an NLRP3 inflammasome inhibitor and may represent a potential therapeutic agent for managing NLRP3-mediated inflammatory disease.