STING is an endoplasmic reticulum (ER)-resident protein that regulates innate immune signaling. Identified through expression cloning, STING contains five transmembrane domains and activates NF-κB and IRF3 pathways to induce type I interferon (IFN) and an antiviral state. STING deficiency renders cells susceptible to negative-stranded viruses like VSV and impairs intracellular B-form DNA and herpesvirus-induced IFNβ, but not TLR pathways. STING interacts with RIG-I and TRAPβ, a component of the translocon complex involved in ER protein translocation. RNAi ablation of TRAPβ or Sec61β inhibits STING's IFNβ-inducing activity. STING is ubiquitously expressed in tissues and resides predominantly in the ER. Overexpression of STING robustly induces IFNβ-Luc, while loss of STING abrogates viral and DNA-induced IFNβ. STING-deficient mice are highly susceptible to VSV infection, confirming its role in innate immune signaling. STING functions downstream of RIG-I and IPS-1, facilitating RIG-I-mediated signaling. STING also interacts with the translocon, suggesting a role in ER-associated innate signaling. STING may detect viral RNA at the ribosome/ER translocon interface and contribute to ER stress responses. STING's interaction with TBK1 and the exocyst complex highlights its role in innate immune signaling. STING is essential for intracellular DNA-induced IFNβ, acting independently of TLR pathways. These findings reveal STING as a critical regulator of innate immune signaling, linking ER translocon function to antiviral responses.STING is an endoplasmic reticulum (ER)-resident protein that regulates innate immune signaling. Identified through expression cloning, STING contains five transmembrane domains and activates NF-κB and IRF3 pathways to induce type I interferon (IFN) and an antiviral state. STING deficiency renders cells susceptible to negative-stranded viruses like VSV and impairs intracellular B-form DNA and herpesvirus-induced IFNβ, but not TLR pathways. STING interacts with RIG-I and TRAPβ, a component of the translocon complex involved in ER protein translocation. RNAi ablation of TRAPβ or Sec61β inhibits STING's IFNβ-inducing activity. STING is ubiquitously expressed in tissues and resides predominantly in the ER. Overexpression of STING robustly induces IFNβ-Luc, while loss of STING abrogates viral and DNA-induced IFNβ. STING-deficient mice are highly susceptible to VSV infection, confirming its role in innate immune signaling. STING functions downstream of RIG-I and IPS-1, facilitating RIG-I-mediated signaling. STING also interacts with the translocon, suggesting a role in ER-associated innate signaling. STING may detect viral RNA at the ribosome/ER translocon interface and contribute to ER stress responses. STING's interaction with TBK1 and the exocyst complex highlights its role in innate immune signaling. STING is essential for intracellular DNA-induced IFNβ, acting independently of TLR pathways. These findings reveal STING as a critical regulator of innate immune signaling, linking ER translocon function to antiviral responses.