Cortisol, the principal corticosteroid in teleosts, is thought to play a key role in the metabolic adjustments critical for regaining homeostasis. However, the target tissue molecular mechanisms involved in this adaptive response are still unclear. In order to elucidate the molecular basis of glucocorticoid receptor (GR)-mediated metabolic re-adjustments, we exposed primary culture of rainbow trout hepatocytes in vitro to cortisol (stressed levels seen in fish) and RU486 (a GR antagonist) either alone or in combination for 24 h and analyzed transcript changes. A low density custom-made rainbow trout cDNA array enriched with endocrine-, metabolic- and stress-related genes was utilized to identify the transcriptome response to GR activation. The microarray results for select genes were further validated using quantitative real-time PCR. Cortisol treatment significantly increased glucose production in hepatocytes and this response was blocked by RU486 confirming GR-mediated corticosteroid signaling. Cortisol also elevated GR transcript levels and this response was abolished by RU486, while both cortisol and RU486 either alone or in combination reduced GR protein content in trout hepatocytes. Cortisol treatment significantly modulated the expression of several genes known to be involved in intermediary metabolism, cellular stress response, reproduction and xenobiotic metabolism. Most of these cortisol-mediated transcript changes were abolished in the presence of RU486, suggesting a key role for GR-specific signaling in the cellular adjustments to defend against stressor insult. Taken together, our results highlight a key role for genomic cortisol signaling in the liver molecular reprogramming that is critical for coping with stress in fish.