ISG15, the first identified ubiquitin-like protein stimulated by type I interferon, has multiple functions in the different vertebrate species and biological processes, such as anti-infection, autophagy, proliferation, cell death, and tumorigenesis.1 ISG15 has also been related to inflammation: human ISG15 deficiency results in necrotizing skin lesions through systemic type I IFN inflammation2; intracellular free ISG15 acts as a negative regulator of IFN-α/β-dependent autoinflammation by keeping USP18 stabilization.3 Despite these findings, the detailed molecular mechanisms how ISG15 regulates inflammation, especially neuroinflammation, remain largely elusive. This study used human microglia (HM cell line) and astrocytes (U87-MG cell line) to explore the molecular mechanisms underlying the association of neuroinflammation with ISG15 and find a negative regulatory mechanism of inflammation by ISG15. ISG15 post-translationally modified Ubc13, inhibiting the binding between Ubc13 and ubiquitin and preventing the K63 polyubiquitination of TRAF6, leading to the NF-κB signaling pathway inactive and then resulting in suppressed expression levels of pro-inflammatory cytokines and NLRP3. Furthermore, ISG15 positively regulated anti-inflammatory cytokines (IL-10, TGF-β, IL-35, IL-37, and IL-38) to prevent the expansion of the inflammatory response. Our finding suggests that ISG15 is a potential therapeutic target for inflammation.