OBJECTIVE—Metabolic stress in obesity induces endothelial inflammation and activation, which initiates adipose tissue inflammation, insulin resistance, and cardiovascular diseases. However, the mechanisms underlying endothelial inflammation induction are not completely understood. Stimulator of interferon genes (STING) is an important molecule in immunity and inflammation. In the present study, we sought to determine the role of STING in palmitic acid–induced endothelial activation/inflammation. APPROACH AND RESULTS—In cultured endothelial cells, palmitic acid treatment activated STING, as indicated by its perinuclear translocation and binding to interferon regulatory factor 3 (IRF3), leading to IRF3 phosphorylation and nuclear translocation. The activated IRF3 bound to the promoter of intercellular adhesion molecule 1 and induced intercellular adhesion molecule-1 expression and monocyte–endothelial cell adhesion. When analyzing the upstream signaling, we found that palmitic acid activated STING by inducing mitochondrial damage. Palmitic acid treatment caused mitochondrial damage and leakage of mitochondrial DNA into the cytosol. Through the cytosolic DNA sensor cyclic GMP-AMP synthase, the mitochondrial damage and leaked cytosolic mitochondrial DNA activated the STING–IRF3 pathway and increased intercellular adhesion molecule-1 expression. In mice with diet-induced obesity, the STING–IRF3 pathway was activated in adipose tissue. However, STING deficiency (Sting) partially prevented diet-induced adipose tissue inflammation, obesity, insulin resistance, and glucose intolerance. CONCLUSIONS—The mitochondrial damage-cyclic GMP-AMP synthase–STING–IRF3 pathway is critically involved in metabolic stress–induced endothelial inflammation. STING may be a potential therapeutic target for preventing cardiovascular diseases and insulin resistance in obese individuals.