Cyclic GMP-AMP synthase (cGAS) binds to microbial and self-DNA in the cytosol and synthesizes cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING) and downstream mediators to elicit an innate immune response. Regulation of cGAS activity is essential for immune homeostasis. Here, we identified the E3 ubiquitin ligase MARCH8 (also known as MARCHF8, c-MIR, and RNF178) as a negative regulator of cGAS-mediated signaling. The immune response to double-stranded DNA was attenuated by overexpression of MARCH8 and enhanced by knockdown or knockout of MARCH8. MARCH8 interacted with the enzymatically active core of cGAS through its conserved RING-CH domain and catalyzed the lysine-63 (K63)-linked polyubiquitylation of cGAS at Lys411. This polyubiquitylation event inhibited the DNA binding ability of cGAS, impaired cGAMP production, and attenuated the downstream innate immune response. Furthermore, March8-deficient mice were less susceptible than their wild-type counterparts to herpes simplex virus 1 (HSV-1) infection. Together, our findings reveal a mechanism underlying the functional regulation of cGAS and the fine-tuning of the innate immune response.