Toll-like receptors (TLRs) initiate a signalling cascade via association with an adaptor molecule, myeloid differentiation factor 88 (MyD88) and/or TIR domain-containing adaptor inducing-IFN-β (Trif), to induce various pro-inflammatory cytokines for microbial eradication. After stimulation of TLR4 with lipopolysaccharide (LPS), both IL-1β and IL-18 are processed, depending on the activation of caspase-1, although its mechanism remains unclear. ASC is an adapter protein possibly involved in the activation of procaspase-1. To unravel the requirement of ASC, we generated Asc−/– mice. Upon stimulation with LPS, Asc−/– macrophages failed in the processing of procaspase-1 and maturation of pro-IL-1β and pro-IL-18, but normally produced other pro-inflammatory cytokines including TNF-α and IL-6. MyD88−/– and Trif−/– macrophages showed normal activation of caspase-1, demonstrating a dispensable role for MyD88 and Trif. After, LPS-challenged Asc−/– mice lacked serum elevation of IL-1β and IL-18. Moreover, the Asc−/– mice exhibited neither acute liver injury nor lethal shock. These results demonstrate critical roles for ASC in the release of IL-1β/IL-18 via activation of caspase-1 and provide new insights into the inflammatory responses for host defence and diseases.