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The present study evaluated the immunophysiological response in Colossoma macropomum fed with different levels of dietary protein in a biofloc system (BFS) and in clear water (CW) and under infection with Aeromonas jandaei. Juvenile tambaqui (9.20 ± 0.23 g) were fed isolipid feed with three levels of crude protein (CP) in the two production systems: BFS24, BFS28 and BFS32 and CW24, CW28 and CW32 with 24, 28 and 32% CP respectively, for 60 days. At the end of the experimental period, the physiological conditions (hematology, biochemistry, hormonal and oxidative stress) of the fish were analyzed. The results of erythrogram, cortisol, glycemia and serum biochemistry (p > 0.05) of the fish did not show significant differences between the breeding systems (BFS and CW) and the different protein levels. Tambaqui raised in the BFS showed monocytosis, thrombocytosis and higher respiratory activity of leukocytes, as well as higher glutathione (GSH) and lower malondialdehyde (MDA) values (p < 0.05). In the bacterial challenge, after induction of aeromonosis, caused by Aeromonas jandaei, greater survival of fish raised in the BFS was observed. The results suggest that, even at lower protein levels, tambaqui maintain physiological homeostasis and, therefore, it may be possible to use up to 24% CP in the diet in the biofloc system. In addition, after the bacterial infection, this system promoted greater immunological resistance in the fish.