NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3) involves in inflammasome complex assembly and innate immunity. Activation of the NLRP3 inflammasome induces conformational alterations in protein complexes, influencing their interactions with other molecules, which in turn affects protein thermal stability. To investigate the proteome-wide thermal stability alterations induced by NLRP3 inflammasome activation, we conducted a comprehensive analysis of meltome dynamics using thermal proteome profiling. Our analysis identified 337 proteins exhibiting alterations in thermal stability upon NLRP3 inflammasome activation. Subsequently, we validated three proteins by the cellular thermal shift assay. Notably, our findings reveal that the majority of these proteins tend to cluster into distinct macromolecular complexes. Furthermore, we identified FAM120A as a novel NLRP3 binding partner, with its suppression enhancing caspase-1 activation and IL-1β release in response to NLRP3 agonist. Collectively, these data provide a comprehensive framework for understanding the mechanisms of NLRP3 inflammasome activation and underscore the utility of thermal proteome profiling in exploring proteome-wide thermal stability changes during signaling transduction.