Abstract Inflammasomes are multimeric protein complexes that are activated through a NOD-like receptor and regulate the proteolytic activation of caspase-1 and cytokines, like IL-1β. The NLRP3 inflammasome is implicated in many human pathologies including infections, autoinflammatory syndromes, chronic inflammation, and metabolic diseases; however, the molecular mechanisms of activation are not fully understood. In this study we show that NLRP3 inflammasome activation requires intracellular copper. A clinically approved copper chelator, tetrathiomolybdate, inhibited the canonical NLRP3 but not the AIM2, NLRC4, and NLRP1 inflammasomes or NF-κB–dependent priming. We demonstrate that NLRP3 inflammasome activation is blocked by removing copper from the active site of superoxide dismutase 1, recapitulating impaired inflammasome function in superoxide dismutase 1–deficient mice. This regulation is specific to macrophages, but not monocytes, both in mice and humans. In vivo, depletion of bioavailable copper resulted in attenuated caspase-1–dependent inflammation and reduced susceptibility to LPS-induced endotoxic shock. Our results indicate that targeting the intracellular copper homeostasis has potential for the treatment of NLRP3-dependent diseases.