An 8-wk feeding trial was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) on growth, body composition, and fatty acid profiles of juvenile Acanthopagrus schlegeli. Three replicate groups of fish (initial mean weight: 8.08 ± 0.09 g, mean ± SD) were fed diets with different levels of n-3 HUFA (0.76%, HUFA0.76; 0.83%, HUFA0.83; 0.90%, HUFA0.90; 0.97%, HUFA0.97; 1.04%, HUFA1.04; 1.12%, HUFA1.12) at 12.9% of total lipid, with a constant eicosapentaenoic acid (EPA, 20:5n-3) to docosahexaenoic acid (DHA, 22:6n-3) ratio of about 2.1. Hepatosomatic index (HSI) and intraperitoneal fat (IPF) ratio were all linearly depressed by dietary n-3 HUFA levels (P < 0.05), and condition factor (CF) was not affected. Adipocyte diameter in IPF decreased with the elevation of dietary n-3 HUFA and significance occurred between group HUFA0.90 and HUFA1.12. Lipid content in dorsal muscle was significantly lowered by dietary n-3 HUFA compared to fish fed diet HUFA0.76. No significance was found in whole fish proximate composition. In liver, dorsal muscle and IPF, ∑SFA, 16:0 or ∑n-3 HUFA were all positively correlated with dietary n-3 HUFA, while DHA to EPA ratios remained constant in 2.68, 2.86, and 3.60, respectively. Fatty acid synthase (FAS, EC 220.127.116.11) activities of all treatments remained constant at first and then were significantly elevated by dietary n-3 HUFA higher than 0.97% (P < 0.05). In contrast, hormone sensitive lipase (HSL, EC 18.104.22.168) changed following an opposite tendency. Quadratic analysis based on weight gain rate (WGR) indicated that dietary n-3 HUFA requirement for juvenile black seabream was 0.94% of the diet in 12.9% lipid diets.