NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome is a component of innate immunity, and is responsible for producing mature IL-1β and -18. Several flavonoids were found to affect inflammasome pathway, but the mechanism of action is still obscure. To elucidate the effects on NLRP3 inflammasome pathway and to determine the structure-activity relationships, NLRP3 inflammasome in differentiated THP-1 cells was activated via treatment with monosodium urate (MSU) crystals. Levels of mature IL-1β, NLRP3 inflammasome components and apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain) (ASC) oligomerization were investigated and the mechanisms of action were also elucidated. Among the 56 flavonoids initially tested, only flavone, 2′,4′-dihhydroxyflavone, 3′,4′-dichloroflavone, 4′,5,7-trihydroxyflavone (apigenin), 3,4′,5,7-tetrahydroxyflavone (kaempferol) and 3,3′,4′,5,7-pentahydroxyflavone (quercetin) significantly inhibited IL-1β production at 10μM. Apigenin, kaempferol and 3′,4′-dichloroflavone inhibited ASC oligomerization without affecting the ASC level in cell lysates. Apigenin also inhibited absent in melanoma 2 (AIM2) inflammasome-related pathway, but not NLR family CARD domain-containing protein 4 (NLRC4) inflammasome activation. The action of apigenin on NLRP3 inflammasome activation is mediated partly via inhibition of phosphorylation of spleen tyrosine kinase/protein tyrosine kinase 2 (Syk/Pyk2) pathway. Furthermore, orally administered apigenin (100mg/kg) strongly reduced the number of neutrophils and monocytes in MSU-induced peritonitis in mice. The present study, for the first time, demonstrated the structure-activity profiles of flavonoids in NLRP3 inflammasome activation and mechanisms of cellular action. Certain flavonoids including apigenin are expected to ameliorate the inflammatory symptoms in autoinflammatory diseases associated with NLRP3 inflammasome activation.