The effects of long-term freshwater acclimatization were investigated in juvenile sea-bass Dicentrarchus labrax to determine whether all sea-bass juveniles are able to live in freshwater and to investigate the physiological basis of a successful adaptation to freshwater. This study particularly focused on the ability of sea-bass to maintain their hydromineral balance in freshwater and on their ion (re)absorbing abilities through the gills and kidneys. Two different responses were recorded after a long-term freshwater acclimatization. (1) Successfully adapted sea-bass displayed standard behavior; their blood osmolality was maintained almost constant after the freshwater challenge, attesting to their efficient hyperosmoregulation. Their branchial and renal Na+/K+-ATPase abundance and activity were high compared to seawater fish due to a high number of branchial ionocytes and to the involvement of the urinary system in active ion reabsorption, producing hypotonic urine. (2) Sea-bass that had not successfully adapted to freshwater were recognized by abnormal schooling behavior. Their blood osmolality was low (30% lower than in the successfully adapted sea-bass), which is a sign of acute osmoregulatory failure. High branchial Na+/K+-ATPase abundance and activity compared to successfully adapted fish were coupled to a proliferation of gill chloride cells, whose ultrastructure did not display pathological signs. The large surface used by the gill chloride cells might negatively interfere with respiratory gas exchanges. In their urinary system, enzyme abundance and activity were low, in accordance with the observed lower density of the kidney tubules. Urine was isotonic to blood in unsuccessfully adapted fish, ruling out any participation of the kidney in hyperosmoregulation. The kidney failure seems to generate a compensatory ion absorption through increased gill activity, but net ion loss through urine seems higher than ion absorption by the gills, leading to lower hyper-osmoregulatory performance and to death.