The inflammasome is a major component of the innate immune system, and its main function is to activate caspase-1, a cysteine protease that promotes inflammation by inducing interleukin-1β (IL-1β) maturation and release into the extracellular milieu. To prevent uncontrolled inflammation, this complex is highly regulated. When it is assembled, the inflammasome is insoluble, which has long precluded the analysis of its interactions with other proteins. Here we used the proximity-dependent biotinylation assay (BioID) to identify proteins associated with caspase-1 during inflammasome activation. Using the BioID in a cell-free system in which the inflammasome had been activated, we found that a caspase-1-biotin ligase fusion protein selectively labeled 111 candidates, including the p62/sequestosome-1 protein (p62). Using co-immunoprecipitation experiments, we demonstrated that p62 interacts with caspase-1. This interaction promoted caspase-1-mediated cleavage of p62 at Asp-329. Mechanistic and functional analyses revealed that caspase-1-mediated cleavage of p62 leads to loss of its interaction with the autophagosomal protein microtubule-associated protein 1 light chain 3 β (LC3B). Strikingly, overexpression of a p62 N-terminal fragment generated upon caspase-1 cleavage decreased IL-1β release, whereas overexpression of p62's C-terminal portion enhanced IL-1β release, by regulating pro-IL1β levels. Overall, the overexpression of both fragments together decreased IL-1β release. Taken together, our results indicate that caspase-1-mediated p62 cleavage plays a complex role in balancing caspase-1-induced inflammation.,© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.