The transition from freshwater to seawater is an extreme physiological challenge for Atlantic salmon smolts. Stressors occurring during downstream migration impair the ability of smolts to maintain ionic/osmotic homeostasis in seawater, however the molecular mechanisms underlying this are not fully understood. We used RNA-seq and measures of whole animal performance to examine the molecular basis of impaired seawater tolerance after an acute aquaculture-related stressor. Smolts were challenged with 24h of seawater before and after exposure to acute netting/confinement stress. RNA-Seq followed by Differential Expression and Weighted Gene Co-expression Network Analysis (WGCNA) was used to quantify the transcriptional response of the gill to netting/confinement stress, seawater, and their interaction. Netting/confinement impaired seawater tolerance, as indicated by increases in plasma osmolality and chloride in stressed smolts as compared to controls. Exposure to netting/confinement stress, seawater, and their interaction resulted in 2717, 1007, and 1390 differentially expressed genes, respectively, indicating a different profile of transcriptional activity in the gill under each condition. WGCNA identified 15 modules (groups of co-expressed genes), 10 of which were correlated to one or more performance traits: glucose, chloride, and osmolality. Two-way ANOVA revealed that 7 modules were significantly affected by stress, 6 by seawater, and 2 by their interaction. These data suggest that acute stressors cause rapid shifts in the magnitude and direction of gene expression in the gills of smolts that persist during early seawater acclimation. Future work will include analyses to explore the functional significance of differentially expressed genes and modules.