Intracellular NOD-like receptors (NLRs) are emerging as critical regulators of innate and adaptive immune responses. Although the NLR family member NLRC5 is functionally defined, the role of NLRC5 in regulating innate immune signaling has been controversial in mammals, and is poorly understood in teleost fish. In the present study, we report the functional characterization of zebrafish NLRC5. The cloned NLRC5 consists of 6435 bp which encodes 1746 amino acids. The N-terminal effector-binding domain of zebrafish NLRC5 is absent which is different from all other human NLRs. Fluorescence microscopy showed that zebrafish NLRC5 is located throughout the entire cell. The higher expression of zebrafish NLRC5 in embryo than in larvae was observed, suggesting the action phase of NLRC5 in zebrafish ontogenetic stages. When the modulation of NLRC5 in pathogen infection was analyzed, it was found that zebrafish NLRC5 was upregulated by both bacterial and viral infection. Overexpression of zebrafish NLRC5 resulted in significant inhibition of SVCV replication in vivo and in vitro, but failed to activate interferon (IFN) promoters and type I IFN signaling pathway. Interestingly, NLRC5 overexpression could activate mhc2dab promoter, and induce the expression of MHC class II genes. All together, these results demonstrate that zebrafish NLRC5 is involved in IFN-independent antiviral response, and also functions as a transcriptional regulator of MHC class II genes.