The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome mediates production of inflammatory mediators, such as IL-1β and IL-18, and as such is implicated in a variety of inflammatory processes, including infection, sepsis, autoinflammatory diseases, and metabolic diseases. The proximal steps in NLRP3 inflammasome activation are not well understood. Here we elucidate a critical role for Ca²⁺ mobilization in activation of the NLRP3 inflammasome by multiple stimuli. We demonstrate that blocking Ca²⁺ mobilization inhibits assembly and activation of the NLRP3 inflammasome complex, and that during ATP stimulation Ca²⁺ signaling is pivotal in promoting mitochondrial damage. C/EPB homologous protein, a transcription factor that can modulate Ca²⁺ release from the endoplasmic reticulum, amplifies NLRP3 inflammasome activation, thus linking endoplasmic reticulum stress to activation of the NLRP3 inflammasome. Our findings support a model for NLRP3 inflammasome activation by Ca²⁺-mediated mitochondrial damage.