Turbot harbor a relatively remarkable ability to adapt to opposing osmotic challenges and are an excellent model species to study the physiological adaptations of flounder associated with osmoregulatory plasticity. The kidney transcriptome of turbot treated 24 h in water of hypo-salinity (salinity 5) and seawater (salinity 30) was sequenced and characterized. In silico analysis indicated that all unigenes had significant hits in seven databases. The functional annotation analysis of the transcriptome showed that the immune system and biological processes associated with digestion, absorption, and metabolism played an important role in the osmoregulation of turbot in response to hypo-salinity. Analysis of biological processes associated with inorganic channels and transporters indicated that mineral absorption and bile secretion contributed to iono-osmoregulation resulting in cell volume regulation and cell phenotypic plasticity. Moreover, we analyzed and predicted the mechanisms of canonical signaling transduction. Biological processes involved in renin secretion, ECM-receptor interaction, adherens junction, and focal adhesion played an important role in the plasticity phenotype in hypo-stress, while the signal transduction network composed of the MAPK signaling pathway and PI3K-Akt signaling pathway with GABAergic synapse, worked in hypo-osmoregulation signal transduction in the turbot. In addition, analysis of the tissue specificity of targeted gene expression using qPCR during salinity stress was carried out. The results showed that the kidney, gill, and spleen were vital regulating organs of osmotic pressure, and the osmoregulation patt of euryhaline fish differed among species.