Smoltification is stressful for salmonids, and cortisol is one of the central endocrine regulators for seawater adaptation. It has been established that cortisol plays both mineralocorticoid and glucocorticoid functions by MR and GR, respectively, since the aldosterone hormone is absent. Recently, investigations have proposed that the 11-deoxycorticosterone (DOC) mineralocorticoid precursor might support cortisol effects, but this mechanism remains unclear. Hence, we assessed the early effects of DOC on rainbow trout pre-smolts, the key smoltification stage, via metabolic and transcriptomic approaches. Thirty-six juveniles (similar to 120 g) were treated for 3 h with DOC (1 mg/kg) and/or mineralocorticoid (eplerenone) or glucocorticoid (mifepristone) receptor antagonists (n = 6 for each group). DOC decreased plasma glucose and pyruvate and increased phosphate and liver glycogen. DOC also downregulated carbohydrate metabolism-related genes in the liver. Finally, gill RNA-seq analysis presented 1660 differentially expressed transcripts in DOC versus vehicle, 1022 for eplerenone + DOC versus DOC and 3324 for mifepristone + DOC versus DOC. The enrichment analysis mainly revealed the upregulation of ion transmembrane transport and carbohydrate metabolism and the downregulation of stress and innate immune responses. This suggests a significant role of DOC in liver carbohydrate metabolism and gill osmoregulation of pre-smolts through both receptors. Hence, this could contribute to improving animal welfare monitoring during smoltification by featuring novel and potential biomarkers.