Aquaculture is one of the world's fastest-growing sectors in food production but with multiple challenges related to animal handling and infections. The disease caused by infectious salmon anemia virus (ISAV) leads to outbreaks of local epidemics, reducing animal welfare, and causing significant economic losses. The composition of feed has shifted from marine ingredients such as fish oil and fish meal towards a more plant-based diet causing reduced levels of eicosapentaenoic acid (EPA). The aim of this study was to investigate whether low or high levels of EPA affect the expression of genes related to the innate immune response 48 h after infection with ISAV. The study includes seven experimental groups: ± ISAV and various levels of EPA up to 200 µM. Analysis of RNA sequencing data showed that more than 3000 genes were affected by ISAV alone (without additional EPA). In cells with increasing levels of EPA, more than 2500 additional genes were differentially expressed. This indicates that high levels of EPA concentration have an independent effect on gene expression in virus-infected cells, not observed at lower levels of EPA. Analyses of enriched biological processes and molecular functions (GO and KEGG analysis) revealed that EPA had a limited impact on the innate immune system alone, but that many processes were affected by EPA when cells were virus infected. Several biological pathways were affected, including protein synthesis (ribosomal transcripts), peroxisome proliferator activated receptor (PPAR) signaling, and ferroptosis. Cells exposed to both increasing concentrations of EPA and virus displayed gene expression patterns indicating increased formation of oxygen radicals and that cell death via ferroptosis was activated. This gene expression pattern was not observed during infection at low EPA levels or when Atlantic salmon kidney (ASK) cells were exposed to the highest EPA level (200 μM) without virus infection. Cell death via ferroptosis may therefore be a mechanism for controlled cell death and thus reduction of virus replication when there are enough polyunsaturated fatty acids (PUFAs) in the membrane.