Increasing evidence indicates that caspase recruitment domain (CARD)‐mediated caspase‐1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)‐1β release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo‐oligomer. Oligomerized CARD16 formed a filament‐like structure with CASP1CARD and a speck with apoptosis‐associated speck‐like protein containing a CARD. A filament‐like structure formed by CARD16 promoted CASP1 filament assembly and IL‐1β release. In contrast, CARD17 did not form a homo‐oligomer or filaments and inhibited CASP1‐dependent IL‐1β release. Mutated CARD16D27G, mimicking the CARD17 amino acid sequence, formed a homo‐oligomer but failed to form a filament‐like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL‐1β release. These results suggest that oligomerized CARD16 promotes CARD‐mediated molecular assembly and CASP1 activation.
