Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH(2)) of ∼100 nm in diameter, but not carboxyl- or nonfunctionalized particles, trigger NLRP3 inflammasome activation and subsequent release of proinflammatory interleukin 1β (IL-1β) by human macrophages. PS-NH(2) induced time-dependent proton accumulation in lysosomes associated with lysosomal destabilization, release of cathepsin B, and damage of the mitochondrial membrane. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein, resulting in a conformational change of the pyrin domain of the NLRP3 protein, as was predicted by molecular modeling. Consequently, this prompted assembly of the NLRP3 inflammasome complex with recruitment and activation of caspase-1, inducing IL-1β release by cleavage of pro-IL-1β. The central role of the NLRP3 inflammasome for cytokine production was confirmed by in vitro knockdown of NLRP3 and of the adaptor protein ASC, confirming that other inflammasomes were not activated by PS-NH(2). The PS-NH(2)-mediated proinflammatory macrophage activation could be antagonized by the radical scavenger N-acetyl-L-cysteine, which prevented mitochondrial damage, caspase-1 activation, and the subsequent release of IL-1β. Our study reveals the molecular mechanism of NLRP3 inflammasome activation by amino-functionalized nanoparticles and suggests a strategy as to how such adverse effects could be antagonized.