Cyclic GMP-AMP synthase (cGAS) is an essential sensor of cytosolic DNA and critically mediates innate immune responses and autoimmunity. Modulating the activity and stability of cGAS provides potential strategies for treating viral or autoimmune diseases. Here, we report that ubiquitin-specific protease 29 (USP29) deubiquitinates and stabilizes cGAS and promotes cellular antiviral responses and autoimmunity. Knockdown or knockout of USP29 severely impairs Herpes simplex virus 1 (HSV-1)- or cytosolic DNA-induced expression of type I interferons (IFNs) and proinflammatory cytokines. Consistently, Usp29m/m mice produce decreased type I IFNs and proinflammatory cytokines after HSV-1 infection and are hypersensitive to HSV-1 infection compared to the wild-type littermates. In addition, genetic ablation of USP29 in Trex1-/- mice eliminated the detectable pathological and molecular autoimmune phenotypes. Mechanistically, USP29 constitutively interacts with cGAS, deconjugates K48-linked polyubiquitin chains from cGAS and stabilizes cGAS in uninfected cells or after HSV-1 infection. Reconstitution of cGAS into Usp29-/- cells fully rescues type I IFN induction and cellular antiviral responses after HSV-1 infection. Our findings thus reveal a critical role of USP29 in the innate antiviral responses against DNA viruses and autoimmune diseases and provide insight into the regulation of cGAS.