Background <p>In teleost fish, adipose tissue serves as the primary site for lipid storage and metabolism regulation, with distinct depots located in visceral (VAT), subcutaneous (SAT), and intramuscular (IMAT) regions. While previous studies have described some structural and functional differences among these depots in rainbow trout (<i>Oncorhynchus mykiss</i>), comprehensive transcriptomic data remain limited. The present study applies high-throughput RNA sequencing to characterize mRNA (RNA-seq) and miRNA (miRNA-seq) transcription across these three major adipose tissue depots, with the aim of exploring depot-specific regulatory patterns, identifying key miRNAs involved in lipid metabolism, and predicting their potential regulatory role.</p> Results <p>Transcriptomic analyses of isolated mature adipocytes from VAT, SAT, and IMAT revealed distinct expression and regulatory profiles. The shared expression of a gene cluster between SAT and IMAT suggested partial overlap in functional traits. Notably, depot-specific miRNAs were detected, indicating unique post-transcriptional modulation depending on adipose location. Gene ontology (GO) enrichment of differentially expressed (DE) mRNAs highlighted functional specializations with VAT being primarily associated with lipid metabolism and immune response, SAT with energy balance and cellular signaling, and IMAT with energy and phospholipid metabolism. Canonical mRNA profile analyses further reinforced these depot different signatures. Although depot-specific miRNAs did not show significant pathway enrichment, DE miRNA biological processes supported depot differential post-transcriptional modulation over previously enriched mRNA pathways. While known mammalian miRNA–mRNA interactions could not be confirmed in rainbow trout in the present study, other potential interactions, with significant impact on cell physiology, were observed. These results underscore the molecular heterogeneity of adipose depots and suggest that miRNAs may selectively shape depot functionality in rainbow trout.</p> Conclusions <p>Transcriptomic analyses suggest that although adipose tissue depots share common roles, certain functions were more prominent in specific depots. VAT seems to be more focused towards lipid storage and immune regulation, with strong miRNA post-transcriptional control. SAT appears to be flexible, maintaining metabolic balance and fat modulation, exhibiting some miRNA regulation. IMAT would be mainly involved in energy metabolism, interacting with muscle, and apparently less dependent on miRNA regulation compared to VAT and SAT. This study enhances the understanding of adipose depots particularities in rainbow trout, and establishes the basis for developing strategies to improve adiposity management on aquaculture fish.</p>