AbstractCytosolic inflammasomes are supramolecular complexes that are formed in response to intracellular pathogens and danger signals. However, as to date, the detailed description of a homotypic caspase recruitment domain (CARD) interaction between NLRP1 and ASC has not been presented. We found the CARD–CARD interaction between purified NLRP1^CARD and ASC^CARD experimentally and the filamentous supramolecular complex formation in an in vitro proteins solution. Moreover, we determined a high-resolution crystal structure of the death domain fold of the human ASC^CARD. Mutational and structural analysis revealed three conserved interfaces of the death domain superfamily (Type I, II, and III), which mediate the assembly of the NLRP1^CARD/ASC^CARD complex. In addition, we validated the role of the three major interfaces of CARDs in assembly and activation of NLRP1 inflammasome in vitro. Our findings suggest a Mosaic model of homotypic CARD interactions for the activation of NLRP1 inflammasome. The Mosaic model provides insights into the mechanisms of inflammasome assembly and signal transduction amplification.