Single-molecule localization microscopy reveals that Stimulator of Interferon Genes (STING) clusters at the trans-Golgi network (TGN) through palmitoylation-dependent accumulation of cholesterol. The clustering of STING enhances its association with TANK-binding kinase 1 (TBK1), which is crucial for the activation of interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB). The study provides quantitative evidence for the role of STING as a scaffold in the STING signaling pathway and reveals the mechanism by which STING palmitoylation facilitates its activation. Additionally, the presence of disease-causative COPA variants increases STING clustering, suggesting a link between STING clustering and immune dysregulation. The results also show that cholesterol in the TGN facilitates STING clustering and that palmitoylation-dependent STING clustering increases the encounter probability of TBK1 and STING, potentially enhancing TBK1 activation.Single-molecule localization microscopy reveals that Stimulator of Interferon Genes (STING) clusters at the trans-Golgi network (TGN) through palmitoylation-dependent accumulation of cholesterol. The clustering of STING enhances its association with TANK-binding kinase 1 (TBK1), which is crucial for the activation of interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB). The study provides quantitative evidence for the role of STING as a scaffold in the STING signaling pathway and reveals the mechanism by which STING palmitoylation facilitates its activation. Additionally, the presence of disease-causative COPA variants increases STING clustering, suggesting a link between STING clustering and immune dysregulation. The results also show that cholesterol in the TGN facilitates STING clustering and that palmitoylation-dependent STING clustering increases the encounter probability of TBK1 and STING, potentially enhancing TBK1 activation.