This study evaluated the supplemental effects of linseed oil (LO) as a substitute for docosahexaenoic acid oil (DHAO) in the diet of juvenile Atlantic bluefin tuna. A control diet (DHA) was formulated to contain 65% enzyme-treated fish meal and 3% of DHAO. A LO diet was formulated to contain 1% LO replacing 1% DHAO in DHA diet. In a feeding trial, 300 juvenile bluefin tuna (initial body weight 1.15 g) were randomly divided into two concrete tanks (70 ton capacity) and fed one of the experimental diets for 13 days. Weight gain was higher in the LO group (519%) than in the control (443%) while survival and protein digestibility were similar between groups. The biological assessment of the tuna digestive organs did not differ between the DHA and LO groups. The fatty acid composition of the carcass showed that α-linolenic acid was only observed in the LO group, and there was no difference in the composition of eicosapentaenoic acid and docosapentaenoic acid between the groups. These results indicate that LO could be a dietary good oil source for Atlantic bluefin tuna without apparent negative effects.