Abstract: The glutamate–glutamine cycle is crucial for neuronal function; however, its role in pathological processes in teleost remains poorly understood. This study employed multi-omics approaches to investigate red-spotted grouper nervous necrosis virus (RGNNV)-induced neurodegeneration, integrating transcriptomic, proteomic, single-cell transcriptomic, and ATAC-seq analyses. Our findings revealed neuroactive ligand–receptor interaction and alanine–aspartate–glutamate metabolism as central pathways in RGNNV pathogenesis, particularly through dysregulation of ionotropic glutamate and gamma-aminobutyric acid (GABA) receptors. Significantly, we established a novel cell communication framework demonstrating that altered signaling within the glulb locus correlates with viral susceptibility. Glutamate accumulation led to dysregulation of glutathione and cysteine metabolism and subsequent p53-mediated ferroptosis, as evidenced by the activation of ferritin heavy polypeptide 1a (FTH1a), glutathione peroxidase 4a (GPX4a), and glutamate–cysteine ligase (GCLC) signals. In addition, RGNNV triggered ferroptosis through ferritinophagy-mediated Fenton reaction in grouper kidney cells. This study provides a robust framework for analyzing cell communication in non-model organisms and illuminates the critical role of glutamate–glutamine cycle dysregulation in RGNNV infection and neurodegeneration. Our findings offer new insights into the nervous necrosis virus pathogenesis in teleost and inform future therapeutic strategies.