Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important adaptor molecules in Toll-like receptor (TLR) signaling pathway. In this study, TRAF6 (PoTRAF6) and TAK1 (PoTAK1) were cloned and characterized in Japanese flounder ( Paralichthys olivaceus). The full-length cDNA sequence of PoTRAF6 is 1953 bp, with an open reading frame (ORF) of 1713 bp encoding a putative protein of 570 amino acids. PoTRAF6 contains one really interesting new gene (RING) domain, two zinc fingers, one coiled-coil region, and one meprin and TRAF homology (MATH) domain, which shows a high similarity to TRAF6s in other species. The full-length PoTAK1 cDNA sequence is 2086 bp, with an ORF of 1728 bp that encodes a putative protein of 575 amino acids. PoTAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The promoter regions of PoTRAF6 and PoTAK1 were also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of these two genes were examined in developmental stages, different tissues, and challenged samples. PoTRAF6 and PoTAK1 were expressed during the whole developmental stages, and the highest expressions were in intestine and heart, respectively. In challenged embryonic cells with LPS, CpG ODN, and poly I:C, the expressions of PoTRAF6 and PoTAK1 were both up-regulated significantly. These results suggest that PoTRAF6 and PoTAK1 play crucial roles in immune responses and may be involved in the developmental process of Japanese flounder.