IL-1beta, a key mediator of inflammation, orchestrates a variety of immune responses by initiating gene expression. Herein, we have cloned and sequenced the IL-1beta in orange-spotted grouper (Epinephelus coioides), produced soluble mature recombinant IL-1beta in Escherichia coli, and characterized its biological properties and downstream signal transduction. The grouper IL-1beta cDNA was 1364bp in length, containing an open reading frame of 765bp. The predicted protein of 254 amino acids revealed the presence of the IL-1 family signature motif and the absence of a conventional ICE cut site. Phylogenetically, the grouper IL-1beta clustered closely with those of teleost belonging to Perciformes and apart from those of mammals. The grouper IL-1beta was constitutively expressed in almost all tissues examined, and was augmented in PBL after the addition of LPS or Poly I:C in vitro. The prokaryotically produced rIL-1beta significantly stimulated the proliferation of grouper head kidney cells, and activated gene expression of IL-1beta and COX-2. Moreover, the rIL-1beta-induced IL-1beta and COX-2 expression were reduced by p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125), respectively. Taken together, the present study indicated that grouper IL-1beta may have an important role in grouper immune system and activate similar downstream cascades as its mammalian counterparts.