The study identifies the CRL5–SPSB3 ubiquitin ligase as a key regulator of nuclear cGAS degradation. cGAS, a sensor of aberrant DNA, is tightly regulated within the nucleus to prevent excessive immune activation. The CRL5–SPSB3 complex targets cGAS for ubiquitylation, marking it for degradation via the proteasome. This process is essential for maintaining cellular homeostasis and preventing inappropriate immune responses. The study reveals that the CRL5–SPSB3 complex recognizes a conserved Asn-Asn (NN) motif at the C-terminus of cGAS, which facilitates its recruitment, ubiquitylation, and degradation. Disrupting this process leads to increased cGAS levels, enhancing type I interferon signaling and providing protection against DNA viruses. The research also shows that cGAS is rapidly recruited to chromosomes during mitosis and is degraded in the nucleus, with its levels fluctuating throughout the cell cycle. The CRL5–SPSB3 complex plays a critical role in controlling cGAS stability, and its dysfunction can lead to heightened immune responses. The structural analysis of the cGAS–SPSB3 complex provides insights into the molecular mechanism of cGAS recognition and degradation. These findings highlight the importance of protein degradation in regulating cGAS function and suggest potential therapeutic targets for modulating cGAS activity in immune-related diseases.The study identifies the CRL5–SPSB3 ubiquitin ligase as a key regulator of nuclear cGAS degradation. cGAS, a sensor of aberrant DNA, is tightly regulated within the nucleus to prevent excessive immune activation. The CRL5–SPSB3 complex targets cGAS for ubiquitylation, marking it for degradation via the proteasome. This process is essential for maintaining cellular homeostasis and preventing inappropriate immune responses. The study reveals that the CRL5–SPSB3 complex recognizes a conserved Asn-Asn (NN) motif at the C-terminus of cGAS, which facilitates its recruitment, ubiquitylation, and degradation. Disrupting this process leads to increased cGAS levels, enhancing type I interferon signaling and providing protection against DNA viruses. The research also shows that cGAS is rapidly recruited to chromosomes during mitosis and is degraded in the nucleus, with its levels fluctuating throughout the cell cycle. The CRL5–SPSB3 complex plays a critical role in controlling cGAS stability, and its dysfunction can lead to heightened immune responses. The structural analysis of the cGAS–SPSB3 complex provides insights into the molecular mechanism of cGAS recognition and degradation. These findings highlight the importance of protein degradation in regulating cGAS function and suggest potential therapeutic targets for modulating cGAS activity in immune-related diseases.