A 40-day feeding trial was carried out to investigate the potential of Gracilaria pulvinata meal as a protein source in formulated diets for barramundi (Lates calcarifer) (initial mean body weight of 28.5 ± 0.5 g). In this regard, five practical diets were prepared using a fishmeal-based diet used as positive control (PC) and blends of soybean meal and fishmeal used as a negative control (NC), in which NC was supplemented with 3 (GL3%), 6 (GL6%) and 9% (GL9%) G. pulvinata. Each dietary treatment was replicated in triplicate. Results of the experiment indicated that growth and feeding performance including specific growth rate, feed conversion, protein efficiency ratios and feed intake in all treatments were not significantly different (P > 0.05). Crude protein, lipid and ash contents of carcass were not markedly altered in all dietary treatments. Serum lysozyme, alternative complement activities, serum immunoglobulin and total protein content significantly decreased in fish fed GL9 diets. Increasing dietary supplementation of G. pulvinata decreased the serum triglycerides and cholesterol when compared to the NC. Gracilaria inclusion levels did not affect intestinal total protease and amylase activities (P < 0.05); however, intestine lipase activity in fish fed GL6 diets was significantly higher than other groups (P <0.05). The results of the present study recommend the inclusion of gracilaria meal up to 3%, without significant negative effects on the growth performance, body composition and health parameters of Lates calcarifer.