NOD-like receptors (NLRs) are a family of intracellular pattern recognition receptors (PRRs) that play critical roles in innate immunity against pathogens infection. NLRC5, the largest member of NLR family, has been characterized as a regulator of innate immunity and MHC class I expression. Alternative splicing of NLRC5 is only reported in human and zebrafish. However, the function of NLRC5 isoforms in the innate immune responses remains unknown. In the present study, we report the functional characterization of zfNLRC5a and zfNLRC5d, two splicing isoforms of zebrafish NLRC5. zfNLRC5a and zfNLRC5d are generated by exon skipping, and whose alternative splicing sites exist in the region of LRRs. Fluorescence microscopy showed that zfNLRC5 isoforms were located throughout the entire cell including nuclear staining. The expression of zfNLRC5 isoform was inducible in response to bacterial and viral infections. During SVCV infection, the in vitro and in vivo studies found that zfNLRC5d overexpression increased protection against viral infection; however zfNLRC5a overexpression had no significant effect on antiviral activity. Interestingly, zfNLRC5 isoforms but not zfNLRC5 were involved in transcriptional regulation of TLRs and NF-kappa B signaling. Overexpression of zfNLRC5 isoforms also contributed to negative regulation of antibacterial immune response, with the decreased expression of nfkbiaa (Mkt). All together, these results firstly demonstrate the function of NLRC5 isoforms in antiviral and antibacterial immune responses both in vitro and in vivo. (C) 2018 Elsevier Ltd. All rights reserved.