Double immunoglobulin interleukin-1 receptor-related molecule (DIGIRR) plays an important role in piscine immune response. To investigate the function of DIGIRR in immune response of large yellow croaker (Larimichthys crocea), the cDNA sequence of digirr was cloned, named Lcdigirr. Transcriptional expression levels of Lcdigirr in different tissues and immune challenged spleen, headkidney, skin, gill, intestine, and liver tissue and LCK cells (a kidney cell line from a large yellow croaker) were determined by real-time fluorescence quantitative reverse transcription PCR (qPCR). Then the recombinant plasmid pTurboGFP-DIGIRR was constructed and transfected into HEK293T cells for subcellular localization analysis. The role of LcDIGIRR in nf-κb activation was determined by transfecting the recombinant plasmid pcDNA3.1-DIGIRR into HEK293T cells using dual luciferase reporter system. Sequence analysis showed that the open reading frame (ORF) of Lcdigirr was 1,575 bp in length, encoding 524 amino acids. The LcDIGIRR protein contained two conserved N-terminal immunoglobin (Ig) domains, a transmembrane region, and a typical C-terminal Toll/IL-1 receptor (TIR) domain. Multiple alignments suggested that DIGIRR was highly conserved among the analyzed species. Phylogenetic analysis showed that LcDIGIRR was clustered with bony fish DIGIRR and closely related to spiny-head croaker (Collichthys lucidus). Transcriptional expression analysis indicated that Lcdigirr was expressed in the examined tissues with the most predominant expression in intestine, followed by liver, head-kidney, and spleen. However, the expression of Lcdigirr in heart was very weak. After challenge with pathogenic Pseudomonas plecoglossicida in vivo and LPS, flagellin and poly (I:C) in vitro, Lcdigirr transcripts in main immune tissues and LCK cells were significantly induced at the early stage after stimulation (P < 0.05). Subcellular localization revealed that LcDIGIRR mainly existed in membrane-cytoplasm. The luciferase activities of NF-κB and MyD88-mediated nf-κb were significantly suppressed by overexpression of LcDIGIRR. These findings indicated that Lcdigirr could negatively regulate nf-κb and MyD88-mediated nf-κb activation. The present study might be helpful for better understanding the function of LcDIGIRR in innate immune signaling transduction of large yellow croaker.