Tumor necrosis factor alpha (TNF-α)-induced protein 3 (TNFAIP3/A20) is an important deubiquitinating enzyme that takes part in homeostasis of immunity induced by TNF-α. During viral and bacterial infection, it plays a crucial role in negative regulation of innate immune responses in mammals. However, this molecule in fish is still poorly understood. In this study, we identified full-length A20 gene of Japanese pufferfish (Fugu) Takifugu rubripes and performed its expression analyses in various tissues and stimulated cells. Total length of the determined Fugu A20 gene spaned 6,229 bp and consisted of 9 exons and 8 introns. The Fugu A20 gene contained a 2,394 bp open reading frame (ORF) that encoded a 797-amino acid residue. In the exon-intron structure of Fugu A20 gene, the intronboundary positions in the region having 7 zinc finger (Zf) domains were different from those of human. Expression analysis of Fugu A20 gene exhibited a higher transcription in thymus, head kidney (HK) and spleen tissues than the others. In HK and spleen cells stimulated with LPS, polyI:C and imiquimod, A20 gene was expressed after up-regulation of TNF-α gene expression. These results together suggest that expression of Fugu A20 gene is associated with bacterial and viral infections.