XL-3158 is an orally active inhibitor of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) with human cGAS IC50 of 11.1 μM and murine cGAS IC50 of 2.19 μM. It binds both allosteric and orthosteric sites, stabilizing the inactive closed conformation of the enzyme, thereby weakening cGAS interaction with DNA. By targeting phase separation, XL-3158 effectively penetrates cells, reduces intracellular cGAS local concentration through inhibition of aggregate formation, and shows no significant cytotoxicity at effective concentrations, making it suitable for subsequent cellular function experiments. XL-3158 overcomes species selectivity barriers, presenting a candidate drug for cGAS-dependent inflammatory diseases.