The transition from freshwater to seawater represents a physiological challenge for Atlantic salmon smolts preparing for downstream migration. Stressors occurring during downstream migration to the ocean impair the ability of smolts to maintain osmotic/ionic homeostasis in seawater. The molecular mechanisms underlying this interaction are not fully understood, especially at the organ level. We combined RNA-Seq with measures of whole-animal homeostasis to examine gene expression dynamics in the gills of smolts associated with impaired seawater tolerance after an aquaculture-related stressor. Smolts were given a 24 h seawater tolerance test before and after exposure to an acute handling/confinement stress. RNA-Seq followed by differential expression and weighted gene correlation network analysis (WGCNA) was used to quantify the transcriptional response of the gill to handling/confinement stress, seawater and their interaction. Exposure to acute stress was associated with a general stress response and impaired osmotic/ionic homeostasis in seawater. We identified gene networks in the gill exhibiting response to acute stress alone, seawater alone, and others exhibiting combined effects of both stress and seawater. Our findings indicate that acute handling/ confinement stress increases the intensity of seawater-related gene expression and suggest that increased investment in mechanisms related to ion transport may be part of a compensatory response to impaired seawater tolerance in smolts.