The cGAS-STING pathway plays a crucial role in anti-tumor responses by activating inflammation and reprogramming the tumor microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to the Golgi, facilitating signaling complex assembly and the induction of interferon and inflammatory cytokines. This study reports that cGAMP stimulation transiently reduces ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification, which facilitates ER membrane curvature and STING trafficking to the Golgi. Two cholesterol-binding motifs in STING are identified and confirmed to contribute to ER-retention of STING. Depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumor model, intratumoral administration of cholesterol depletion therapy potentiated STING-dependent anti-tumor responses, which, in combination with anti-PD-1 antibodies, promoted tumor remission. The study demonstrates that ER cholesterol sets a threshold for STING signaling through cholesterol-binding motifs in STING and proposes that this could be exploited for cancer immunotherapy.The cGAS-STING pathway plays a crucial role in anti-tumor responses by activating inflammation and reprogramming the tumor microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to the Golgi, facilitating signaling complex assembly and the induction of interferon and inflammatory cytokines. This study reports that cGAMP stimulation transiently reduces ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification, which facilitates ER membrane curvature and STING trafficking to the Golgi. Two cholesterol-binding motifs in STING are identified and confirmed to contribute to ER-retention of STING. Depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumor model, intratumoral administration of cholesterol depletion therapy potentiated STING-dependent anti-tumor responses, which, in combination with anti-PD-1 antibodies, promoted tumor remission. The study demonstrates that ER cholesterol sets a threshold for STING signaling through cholesterol-binding motifs in STING and proposes that this could be exploited for cancer immunotherapy.