Cytosolic inflammasome protein complexes sense pathogen and sterile danger molecules to activate and induce the secretion of the potent proinflammatory cytokine interleukin-1β (IL-1β). The importance of IL-1β is underscored by clinical trials implicating its excessive activation in hereditary autoinflammatory syndromes, and common conditions such as gout, cancer and cardiovascular disease. However, there remains a fundamental knowledge gap in our understanding of how IL-1β regulated. Here we have used mass-spectrometry to investigate the post-translational regulation of IL-1β. We found that IL-1β is both phosphorylated and ubiquitylated on conserved target residues. Mutation of a single conserved IL-1β ubiquitylation site by CRISPR gene editing revealed how the ubiquitylation of IL-1β regulates its activation by inflammasome protein complexes, both in vitro and in inflammatory disease models in vivo. Moreover, we observed that distinct innate immune stimuli trigger IL-1β ubiquitylation to impact its signalling. Therefore, the post-translational ubiquitylation of IL-1β is likely to influence inflammatory disease pathogenesis.