Abstract: Yeast nucleotides are known to modulate host immunity and gut microbiota. In teleosts, the intestinal mucosa represents a principal portal of viral entry, compromising barrier integrity, yet the mechanisms by which yeast nucleotides potentiate antiviral defenses remain to be elucidated. Herein, this study performed an eight-week feeding trial of coho salmon with graded yeast nucleotide levels (0, 125, 250, 500, and 1000 mg/kg), followed by intraperitoneal IHNV challenge with sampling at four days post-infection, and an in vitro assessment of intestinal mucus from the control and 500 mg/kg groups co-incubated with EPC cells and IHNV to evaluate antiviral efficacy. Coho salmon showed a biphasic growth response to dietary yeast nucleotides, with the 500 mg/kg group achieving the highest growth among all treatments. Yeast nucleotide enhanced intestinal tight junction integrity by upregulating proteins, such as Occludin, and potentiated mucosal immunity via modulation of NF-κB p65. Notably, yeast nucleotides reshaped gut microbiota and were associated with changes in lipid metabolism and increased levels of bioactive metabolites, with taxa such as Romboutsia, Bacillus, Turicibacter and Clostridium sensu stricto 1 showing significant correlations with these metabolic and immune parameters, although direct functional roles remain to be confirmed. Upon IHNV challenge, the 500 mg/kg group demonstrated significantly reduced cumulative mortality and ameliorated virus-induced disruption of intestinal barrier function compared to the control group. Finally, intestinal mucus from 500 mg/kg yeast nucleotides-fed fish conferred antiviral protection in vitro by upregulating host antiviral gene expression in EPC cells. These findings highlight dietary yeast nucleotides as key modulators of antiviral defense and intestinal barrier integrity potentially through microbiota-associated lipid metabolism and bioactive metabolite profiles, while acknowledging that further functional studies are required to establish causality, offering promising nutritional strategies against virus‐induced gut injury.