Interleukin-1β (IL-1β) is a well-characterized cytokine that plays key roles in cellular responses to infection, inflammation, and immunological challenges in mammals. In this study, we identified and analyzed a grass carp (Ctenopharyngodon idella) ortholog of IL-1β (gcIL-1β), examined its expression patterns in various tissues in both healthy and lipopolysaccharide (LPS)-stimulated specimens, and evaluated its proinflammatory activities. The gcIL-1β gene consists of seven exons and six introns. The full-length cDNA sequence contains an open reading frame of 813 nucleotides. The deduced amino acid sequence exhibits a characteristic IL-1 signature but lacks the typical IL-1β converting enzyme cleavage site that is conserved in mammals. In the phylogenetic tree, IL-1βs from grass carp and other members of the Cyprinidae family clustered into a single group. Expression pattern analysis revealed that gcIL-1β is constitutively expressed in all 11 tissues examined, and LPS stimulation leads to significant up-regulation in muscle, liver, intestine, skin, trunk kidney, head kidney, and gill. Recombinant grass carp IL-1β (rgcIL-1β) was generated prokaryotically as a fusion protein of Trx-rgcIL-1β. An anti-rgcIL-1β polyclonal antibody (rgcIL-1β pAb) was raised in mice against the purified Trx-rgcIL-1β. Western blot analysis confirmed that rgcIL-1β pAb reacted specifically with gcIL-1β in C. idella kidney (CIK) cells. Quantitative real-time PCR data indicated that intestinal mRNA expression levels of endogenous IL-1β, IL-1R2, and TNF-α were significantly up-regulated following Trx-rgcIL-1β exposure. The inhibitory activities of rgcIL-1β pAb against the inflammatory response were confirmed in a model of Aeromonas hydrophila-induced intestinal inflammation. Our immunohistochemical study revealed that the degree and intensity of inflammatory cell infiltration are fully consistent with the observed mRNA expression patterns of these key inflammatory genes. Taken together, these data suggest that gcIL-1β plays a critical role in the proinflammatory response in the grass carp intestine.