NOD-like receptors (NLRs) are members of pattern recognition receptors (PRRs) recognized intracellular pathogens. Here, we identified a type of NLR with a CARD domain (NLRC5) in Japanese flounder, Paralichthys olivaceus (JfNLRC5). The coding sequence JfNLRC5 is 5529 bp long and encodes a protein of 1842 deduced amino acid residues. JfNLRC5 transcripts were highly detected in gills, intestine and spleen of healthy fish. In Japanese flounder stimulated with poly (I:C), JfNLRC5 was significantly up-regulated after 24 h at 15 °C and after 3 h at 25 °C. Expression of JfNLRC5 was up-regulated by formalin-killed Edwardsiella tarda but not by formalin-killed Streptococcus iniae. These findings suggest that JfNLRC5 is involved in fish immune response against viral and Gram-negative bacterial infections.