Macrophage migration inhibitory factor (MIF) is a pro-inflammatory molecule, first identified as a cytokine, that exerts multiple effects on immune cells, as well as having functions outside of the immune system. Importantly, MIF is reported to play a role in the pathology of a number of diseases, including inflammatory arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease and a number of cancers. However, little is known about how MIF actually contributes to these pathologies. Similarly, while it has been demonstrated that MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6 and IL-1 family cytokines, how it does so is not clear. Here, we have shown that inhibition of MIF specifically regulates the NLRP3-dependent release of IL-1α, IL-1β and IL-18. Moreover, MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 inflammasome assembly and activation. Further, we have demonstrated that MIF interacts with NLRP3, indicating a specific role for MIF in inflammasome activation/assembly that is likely independent of its proposed role as a cytokine. These data advance our understanding of how MIF regulates inflammation and open up the possibility of targeting MIF as a means to regulate NLRP3 inflammasome activation.