Abstract Salinity is a key physicochemical factor regulating essential biological processes in crustaceans (e.g., growth, development, reproduction), and its most direct effect is on osmoregulation. Palaemon carinicauda exhibits remarkable euryhaline tolerance, making it ideal for studying crustacean osmoregulation. To elucidate the molecular basis of its long-term freshwater adaptation, this study performed transcriptomic profiling of gill tissues from seventh-generation freshwater-adapted (FW, 0.8 ) and seawater control (SW, 25 ) groups. A total of 229.06 million high-quality reads (5.62 Gb) were generated, mapping to the reference genome at an average rate of 79.81%. Principal component analysis showed distinct transcriptomic separation. Differential alternative splicing events were dominated by skipped exons, while 3449 differentially expressed genes (DEGs) were identified, including 2794 upregulated and 655 downregulated genes in FW. Among these, 34 osmoregulation-related DEGs were categorized into six functional modules including ion transport pumps, ion channels, solute carrier families, organic osmolyte metabolism-related genes, transmembrane sensors, and pH/osmotic balance-associated functional genes. GO analysis highlighted significant enrichment of DEGs in hydrolase activity, mesenchyme migration, extracellular space, and cytoskeleton functions, with the highest numbers linked to plasma membrane and ATP binding, while KEGG analysis identified important pathways including proximal tubule bicarbonate reclamation, mineral absorption, focal adhesion, MAPK signalling pathway, and ECM-receptor interaction — findings corroborated by GSEA. qRT-PCR validation of 11 DEGs confirmed their consistent expression trends with RNA-seq data. These findings clarify the molecular basis of freshwater adaptation in P. carinicauda and provide insights for breeding low-salinity-tolerant varieties for saline-alkaline aquaculture.