Interferon regulatory factor 2 (IRF-2) is a multifunctional transcription factor which exhibits both transcriptional activating and repressing activities in the IRF family. In this study, we report an IRF-2 gene isolated from orange-spotted grouper (Epinephelus coioides). The 1854 bp full-length cDNA sequence of EcIRF-2 has been cloned, encoding a putative peptide of 336 amino acids which is highly consistent with the feature of IRF family members. The genomic fragment of EcIRF-2 contains nine exons and eight introns, spanning over approximate 8.8 kb. The expression of EcIRF-2 gene was detected in various tissues of healthy orange-spotted grouper and in four tissues after being challenged with poly I:C or LPS. EcIRF-2 gene is ubiquitously expressed in various healthy fish tissues and is up-regulated in vivo in response to poly I:C or LPS. Subcellular localization analysis of EcIRF-2 suggests it is an intranuclear protein in the fish cells. We believe this research is the first report of fish IRF-2 protein localization. The results in this research establish the base for further study of function mechanism of IRF family members in orange-spotted grouper.