ISG15 deficiency in humans leads to a failure to maintain adequate levels of USP18, triggering an increase in type I interferon production and signalling, and promoting auto-inflammatory disease. Xianqin Zhang et al. show that intracellular ISG15, an interferon-stimulated ubiquitin-like molecule, functions as negative regulator of type I interferon. ISG15 deficiency in humans leads to a failure to maintain adequate levels of USP18, triggering an increase in type I interferon production and signalling, and promoting auto-inflammatory disease. This work suggests that the primary role for ISG15 in humans is not antiviral — a possibility much studied in the past — but is to prevent IFN-α/β-dependent auto-inflammation. Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice1,2,3,4. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viral diseases5. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi–Goutières syndrome and spondyloenchondrodysplasia6,7,8,9. We further show that an absence of intracellular ISG15 in the patients’ cells prevents the accumulation of USP1810,11, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation.