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Simple Summary: Aquaculture produces fish that are then marketed to the population, but the type of production used today generates stress in fish. Constant stress, which occurs through the hormone cortisol, negatively affects the seawater adaptation of juvenile fish, which is detrimental to the aquaculture process. For many years, it was thought that only cortisol influenced physiological responses in fish. However, in recent years, the cortisol intermediary 11-deoxycorticosterone (DOC) has been considered a complementary stress-related hormone in fish. Given the above, this work aims to unravel whether DOC is involved in the regulation of early physiological and transcriptional responses in fish gills. To ascertain this, juvenile rainbow trout were injected with DOC and/or pharmacological inhibitors of corticosteroid receptors. Then, the physiological parameters and RNA sequencing of the gills were analyzed. The experiment concluded that DOC is important during stress and that it is vital for the balance between minerals and water in fish. The results of this work will help us understand the other effects of DOC in fish, as well as help improve the monitoring of animal welfare in the aquaculture industry through the incorporation of novel and potential stress molecular biomarkers. In aquaculture, stress can negatively affect fish growth. For years, the cortisol hormone has been thought to play both glucocorticoid and mineralocorticoid functions. Nevertheless, recent research has suggested that 11-deoxycorticosterone (DOC) released during stress could contribute to cortisol actions, though this process is still misunderstood. Here, we evaluated the DOC effects on physiological and early transcriptional responses by RNA-seq. Juvenile rainbow trout were treated with DOC and/or glucocorticoids (mifepristone) or mineralocorticoid (eplerenone) receptor antagonists. Subsequently, plasma was collected, and cDNA libraries were generated from the gills of vehicle (control), DOC, mifepristone, mifepristone with DOC, eplerenone, and eplerenone with DOC groups. Calcium and phosphate levels in plasma were changed. Results revealed 914 differentially expressed transcripts (DETs) induced by DOC compared with control, mainly associated with sodium ion transmembrane transport, gluconeogenesis, negative regulation of transmembrane transport, and activation of innate immune response. DOC versus eplerenone with DOC comparison displayed 444 DETs related to cell-cell junction organization, canonical glycolysis, positive regulation of immune response, and potassium ion transport. Conversely, no DETs were detected in DOC versus mifepristone with DOC comparison. These data suggest that DOC has a relevant role in gill stress response and ion transport, which is differentially regulated by mineralocorticoid receptors. [ABSTRACT FROM AUTHOR]