This study was conducted to determine the protein requirement of ayu (Plecoglossus altivelis). Two replicate groups of fish initially averaging 6.6 g were fed the five isocaloric diets containing different protein level from 29% to 57% in a flow-through freshwater system for 25 days. White fish meal was used as a sole protein source. Weight gain and feed efficiency of fish increased significantly with dietary protein level up to 43% (P＜0.05) with no additional response above this level. Protein and lipid retention, moisture, protein and lipid contents of body were not affected by dietary protein levels (P>0.05). Daily protein intake increased significantly with dietary protein level, whereas protein efficiency ratio of fish fed the 57% dietary protein decreased (P<0.05). The data obtained in this study indicate that a 43% dietary protein level could be recommended for the optimum growth of ayu.