In order to investigate the relationships among the C-Myc expression, glutamine metabolism and replication of NNV. First of all, the C-Myc gene (GF-1-C-Myc) from Epinephelus coioides fin cell (grouper fin cells, GF-1) was cloned. The full length of GF-1-C-Myc gene cDNA was 814bp with 285bp ORF, encoding 95 amino acid (aa) with leucine zipper domain and helix-ring-helix (HLH) domain. GF-1-C-Myc protein was expressed and purified, and its polyclonal antibody was generated. The expressions of GF-1-C-Myc gene and the replication of NNV were monitored by real-time quantitative PCR (qRT-PCR) and immunoblotting (WB). The results showed that lack of glutamine could inhibit both the expression of GF-1-C-Myc gene and replication of NNV, while additional glutamine could promote both the expression of GF-1-C-Myc gene and replication of NNV. In addition, the expression of GF-1-C-Myc gene was up-regulated in GF-1 cells infected with NNV, and the glutamine in the medium was significantly consumed. Taken together, GF-1-C-Myc gene was involved in the regulation of glutamine metabolism in the cell, subsequently facilitated the replication of NNV. Our results will shed a new light on the prevention and control of NNV infection.