Abstract The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, which is composed of NLRP3, the adaptor protein ASC, and caspase-1, is closely linked to the pathogenesis of metabolic diseases. We investigated whether caffeic acid phenethyl ester (CAPE), an anti-inflammatory phytochemical, could modulate the activation of NLRP3 inflammasome and have beneficial effects on the related disease such as acute gout. In primary mouse macrophages, CAPE blocked NLRP3 inflammasome activation induced by monosodium uric acid (MSU) crystals. In a mouse air pouch inflammation model and an acute gout model, oral administration of CAPE suppressed MSU-induced caspase-1 activation and interleukin (IL)-1b production, correlating with the attenuation of inflammatory symptoms. CAPE inhibited inflammasome activation induced by ASC, but not NLRP3 nor caspase-1. CAPE directly associated with ASC, resulting in the blockade of NLRP3-ASC interaction. These results demonstrate a novel inhibitory mechanism by which small molecules regulate NLRP3 inflammasome targeting ASC, suggesting the therapeutic strategy for NLRP3-related inflammatory diseases.