IL-33/IL-1F11 is a new member of the IL-1 family ligand and provokes T helper-type immune responses. IL-33 is the ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that triggers nuclear factor-κ light chain enhancer of activated B cells (NF-κB) and MAPK signaling. We discovered a novel short splice variant of IL-33 that was termed spIL-33. The new spIL-33 lacks exon 3 containing a proposed caspase-1 cleavage site. We isolated spIL-33 cDNA from the Huh7 human hepatocarcinoma cell line and expressed the recombinant spIL-33 protein in Escherichia coli. The recombinant spIL-33 and pro-IL-33 were not cleaved by caspase-1, unlike IL-18 (IL-1F4). The recombinant spIL-33 was constitutively active, and spIL-33-induced inflammatory cytokine production was caspase-1-independent in HMC-1 and Raw 264.7 cells. The recombinant spIL-33 induced the phosphorylation of IL-1 receptor-associated kinase (IRAK1), NF-κB, p38 MAPK, p44/42 MAPK, and JNK in a time- and dose-dependent manner. Anti-ST2 monoclonal antibody specifically blocked the spIL-33-induced cytokine production. In this study, we identified and characterized a new IL-33 splice variant, which was a constitutively active IL-33 isoform. The existence of constitutively active spIL-33 suggests that the biological activity of IL-33 could be triggered by diverse stimulations during immune responses. Further investigation of the spIL-33 expression pattern may contribute to understanding the involvement of IL-33 in inflammatory disorders.