Simple SummaryGhrelin is a peptide hormone that regulates food intake and energy balance, but also acts as a cytokine in many species to modulate various immune activities. Multiple ghrelin isoforms were discovered in rainbow trout, but the roles of most isoforms are undefined. This study examined the modulation of immune genes by two major ghrelin variants; the originally identified isoform and its three amino acids truncated isoform. After treating primary cells from rainbow trout head kidney with the two ghrelin analogs, the expression patterns of selected immune-related genes were profiled over time by reverse transcriptase-coupled qPCR. The results suggest that the two ghrelin isoforms exerted divergent modulatory effects on various genes at different time points. These results provide further insight into the direct actions of ghrelin as a critical component of the rainbow trout immune system.AbstractGhrelin is a peptide hormone/cytokine that regulates metabolic processes and plays essential roles in the immune system. To evaluate the immunomodulatory actions of ghrelin isoforms in rainbow trout (RT), an in vitro model was utilized with primary cells isolated from fish head kidney (HKD). These RT-HKD cells were treated with synthetic rainbow trout ghrelin and its truncated isoform, desVRQ-ghrelin, over time (0, 2, 4, and 24 h). Reverse transcriptase-coupled qPCR was used to measure the differential expression patterns of genes relevant to various immune processes and genes of antimicrobial peptides. Ghrelin isoform treatments resulted in functional perturbations that displayed overlapping and divergent patterns of gene expression. The differing actions between the two ghrelin isoforms on various assessed genes, and at differing time points, suggested that the two analogs may activate unique pathways, thereby eliciting distinct responses in fish immunity.