Mammalian cylindromatosis (CYLD) has been identified as a tumor suppressor and participates in innate immune signaling transduction in various cell types through negative regulation of NF-κB activation by deubiquitinating TRAF6 and NEMO. To investigate the role of CYLD in the immune response of large yellow croaker (Larimichthys crocea), the cDNA sequence of CYLD was cloned and identified, named LcCYLD. Gene expression profile was detected by real-time fluorescence quantitative PCR. Then the recombinant plasmid pTurboGFP-CYLD was constructed for subcellular localization and transfected into HEK293T cells. For further understanding of the function of CYLD in innate immune, the recombinant overexpression vector pcDNA3.1-CYLD was constructed and transfected into cells, and the activation capability of NF-κB, proinflammatory factors TNF-α and IL-1β were detected. Sequence analysis showed that the ORF of LcCYLD contained 2 754 bp, encoding 917 amino acids. The putative LcCYLD protein contained three conservative N-terminal CAP_GLY domain, a phosphorylation region, and a typical C-terminal UCH domain. Multiple alignments showed that CYLD was highly conserved among the analyzed species. Phylogenetic analysis showed that LcCYLD was clustered with bony fish and closely related to striped bass (Morone saxatilis). Gene expression analysis indicated that LcCYLD expressed in most examined tissues with the most predominant expression in the brain, followed by blood and intestine. However, the expression levels in other tissues are very weak. LPS and poly I:C stimulation significantly induced the transcriptional expression of LcCYLD. Subcellular localization showed that LcCYLD expressed both in cytoplasm and nuclease. Overexpression of LcCYLD could significantly inhibit the immuno-activation of NF-κB and proinflammation of cytokines TNF-α and IL-1β after LPS and poly I:C challenge. These findings suggested that LcCYLD could negatively regulate NF-κB activation. The present study might be helpful for better understanding the function of LcCYLD in innate immune signaling transduction of L. crocea.