A six-week feeding trial was conducted to determine the effect of butyric acid (BA) inclusion in diet on performance of Barramundi (Lates calcarifer). Fish (12.0 ± 0.2 g) were fed with four experimental diets contained 0.0 (control), 2.5, 5.0 and 10.0 g BA kg-1 in triplicate. Fish fed on 5 and 10 g BA kg-1 diets had higher growth and total alkaline protease and lipase activities than the other treatments. Fillet protein content in BA-supplemented groups was higher than the control. Liver catalase activity was highest in 2.5 g BA kg-1 group compared to other groups. The values of liver superoxide dismutase activity and serum total protein concentration in 5 and 10 g BA kg-1 treatments were higher than those in the control (P < 0.05). Moreover, the highest and the lowest level of serum lysozyme activity were noticed in the 10 g BA kg-1 and the control groups, respectively (P < 0.05). Also, the highest respiratory burst activity was observed in fish fed 5 g BA kg-1 feed. Supplementing diet with 2.5 or 5 g BA kg-1 resulted in higher serum hemolytic activity in fish compared with the other groups. The number of red and white blood cells increased in fish fed BA-supplemented diets compared to the control (P < 0.05). In conclusion, the results of this study showed inclusion of BA at 5 g kg-1 diet in L. calcarifer juveniles improved growth and upgraded the general health condition by enhancing fish antioxidant enzymes activities and hemato-immunological responses.