The cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthetase (cGAS)–stimulator of interferon genes (STING) pathway, comprising the DNA sensor cGAS, the second messenger cyclic GMP–AMP (cGAMP), and the endoplasmic reticulum (ER) adaptor protein STING, detects cytoplasmic double-stranded DNA (dsDNA) to trigger type I-interferon responses for host defense against pathogens. Previous studies defined a model for the allosteric activation of cGAS by DNA-binding, but recent work reveals other layers of mechanisms to regulate cGAS activation such as the phase condensation and metal ions, especially the discovery of Mn2+ as a cGAS activator. Activation of the 2′3′-cGAMP sensor STING requires translocating from the ER to the Golgi apparatus. The sulfated glycosaminoglycans at the Golgi are found to be the second STING ligand promoting STING oligomerization and activation in addition to 2′3′-cGAMP, while surpassed levels of 2′3′-cGAMP induce ER-located STING to form a highly organized ER membranous condensate named STING phase-separator to restrain STING activation. Here, we summarize recent advances in the regulation of cGAS–STING activation and their implications in physiological or pathological conditions, particularly focusing on the emerging complexity of the regulation.