It is generally considered that fish respond to dietary energy densities on a consistent basis irrespective of what macronutrient source the dietary energy originates from. To test this assumption, two experiments were undertaken to establish the different roles of protein, lipid and starch as energy sources in underpinning nutritional bioenergetics in juvenile barramundi, Lates calcarifer. To do this, a range of ingredients were evaluated for their digestible protein (DP) and digestible energy (DE) value. Following this, a series of diets were formulated to an equivalent DE basis, and observed a minimum DP:DE ratio required for fish of 80 g. However, in each of the diets the proportion of DE available from protein, lipid or starch was varied to bias the contribution of each macronutrient on the origin that DE when fed to the fish. Growth of fish fed the protein diet was better than those fed the lipid diet, which was better than those fed the starch diet. Feed intake was lower in the protein diet than the lipid diet, and both were lower than the starch diet. Feed conversion was most efficient in the protein diet fed fish, which was better than the lipid diet fed fish, which was better than the starch diet fed fish. Whole fish composition varied among treatments, with differences observed in the dry matter composition, whole body lipid and gastrointestinal tract lipid content. Typically, lipid and dry matter composition were in synchrony and were usually higher in the starch fed fish and lower in the lipid fed fish. When flux of protein, lipid and energy was assessed in terms of deposition efficiencies, some significant differences were observed. Protein deposition efficiency was relatively conservative, but ranged from 33% in the starch diet fed fish to 41% in the lipid diet fed fish. Lipid deposition efficiency was more dramatic; ranging from 40% in the lipid diet to 182% in the starch diet. Energy deposition efficiency was relatively conservative among treatments, ranging from 50% to 56% efficient. Overall, the results from this study show that there is a clear hierarchy in preference for energy substrates by juvenile barramundi, such that protein > lipid > starch.