This study evaluated the effects of replacing fish oil (FO) with beef tallow (BT) in juvenile olive flounder, Paralichthys olivaceus (3.93 ± 0.07 g), over 8 weeks. Seven diets, consisting of 0, 25, 50, 75, and 100% replacement of FO with BT and 63 and 75.9% replacement supplemented with docosahexaenoic acid (DHA) at 0.9 and 1.1% of 100 g diet, respectively, were distributed into 21 tanks in a semi-recirculation system with 15 fish per tank. The replacement of FO with BT at the given levels showed no significant changes (p > .05) in growth performance and whole-body proximate composition. Fish fed the diet of 75.9% replacement with 1.1% DHA supplementation showed the best performance in these measurements among the treatments. Plasma glutamic pyruvic transaminase, cholesterol, highand low-density lipoproteins, and total protein were not significantly influenced by the replacement of FO with BT. The FO replacement generally resulted in a reduction of eicosapentaenoic acid, DHA, and n-3/n-6 polyunsaturated fatty acid ratio in the whole body, whereas the DHA supplementation Seunghyung Lee and Frolan A. Aya contributed equally to this work.