AbstractInnate immune system is armed by several lines of pattern recognition receptors to sense various viral infection and to initiate antiviral immune response. This process is under a tight control and the negative feedback induced by infection and/or inflammation is critical to maintain immune homoeostasis and to prevent autoimmune disorders, however, the molecular mechanism is not fully understood. Here we report TRIM29, a ubiquitin E3 ligase, functions as an inducible negative regulator of innate immune response triggered by DNA virus and cytosolic DNA. DNA virus and cytosolic DNA stimulation induce TRIM29 expression robustly in macrophages and dendritic cells, although the basal level of TRIM29 is undetectable in those cells. TRIM29 deficiency elevates IFN-I and proinflammatory cytokine production upon viral DNA and cytosolic dsDNA stimulation. Consistently, in vivo experiments show that TRIM29-deficient mice are more resistant to HSV-1 infection than WT controls, indicated by better survival rate and reduced viral load in organs. Mechanism studies suggest that STING–TBK1–IRF3 signaling pathway in TRIM29 KO cells is significantly enhanced and the degradation of STING is impaired. Furthermore, we identify that TRIM29 targets STING for K48 ubiquitination and degradation. This study reveals TRIM29 as a crucial negative regulator in immune response to DNA virus and cytosolic DNA, preventing potential damage caused by overcommitted immune responses.