Tuna muscle by-product powder (TMP), obtained from residues of tuna muscle, was replaced with fishmeal to evaluate its quality, substitution level and phosphorus loading for juvenile Japanese flounder. TMP was substituted for fishmeal on a crude protein basis with levels of 25%, 50%, 75% and 100%, respectively. The feeding trial continued for 40days. Weight gain, specific growth rate and feed intake of fish were not significantly different among the fish fed the control (0% TMP inclusion), 25% and 50% TMP containing diets. However, these parameters significantly (P<0.05) decreased in fish fed diets containing TMP at 75%, 100% with or without phosphorus supplements compared to those fed diets containing TMP at 0%, 25% and 50%. The dietary free histidine level increased with increasing TMP levels while other free amino acids decreased. Negative correlation (R 2 =0.978) was observed between dietary free histidine level and feed intake. Phosphorus (P) loading markedly decreased with increasing TMP level in the diets. The present study demonstrated that TMP is a promising feed ingredient to reduce P discharge into the environment, and could replace 50% of fishmeal protein without reduction of growth performances, resulting in about 50% lower P loading than the control group.