The study investigates the regulation of nuclear cGAS, a key sensor of DNA in the nucleus, which is essential for innate immune responses. The authors identify the ubiquitin proteasomal system (UPS) as the primary mechanism for degrading nuclear cGAS in cycling cells. They discover that SPSB3, a substrate receptor, associates with the Cullin–RING ubiquitin ligase 5 (CRL5) complex to target nuclear cGAS for ubiquitylation and subsequent degradation. Cryo-electron microscopy reveals a conserved Asn-Asn (NN) degron motif in the C-terminal region of cGAS that directs SPSB3 recruitment and ubiquitylation. Interfering with SPSB3-regulated nuclear cGAS degradation enhances type I interferon signaling and protects cells from DNA virus infection. The findings provide insights into the regulation of cGAS in the nucleus and suggest therapeutic opportunities targeting this pathway.The study investigates the regulation of nuclear cGAS, a key sensor of DNA in the nucleus, which is essential for innate immune responses. The authors identify the ubiquitin proteasomal system (UPS) as the primary mechanism for degrading nuclear cGAS in cycling cells. They discover that SPSB3, a substrate receptor, associates with the Cullin–RING ubiquitin ligase 5 (CRL5) complex to target nuclear cGAS for ubiquitylation and subsequent degradation. Cryo-electron microscopy reveals a conserved Asn-Asn (NN) degron motif in the C-terminal region of cGAS that directs SPSB3 recruitment and ubiquitylation. Interfering with SPSB3-regulated nuclear cGAS degradation enhances type I interferon signaling and protects cells from DNA virus infection. The findings provide insights into the regulation of cGAS in the nucleus and suggest therapeutic opportunities targeting this pathway.