Simple SummaryAlthough fish meal is considered the main animal protein source in fish diets, its high cost and unavailability limit its use in aquafeed. Recently, the search for other high-quality replacers of fish meal in aquatic feeds is being carried out with increased attentiveness. However, very few investigations have been performed to assess the possible use of whey protein concentrates (WPC) in Nile tilapia feeds. Five replacement percentages of fish meal with WPC (0%, 13.8%, 27.7%, 41.6%, and 55.5%) were assessed. WPC could replace the fish meal in Nile tilapia diets up to 27.7%, with improving the gut health, the total weight of survival fish, and immune status of fish challenged with Aeromonas hydrophila. High inclusion levels of WPC are not recommended in fish diets, since they negatively affected the intestinal and liver tissues and increased the level of cellular apoptosis, as indicated by the increased caspase 3 activity.The present study was conducted to assess the effect of replacing fish meal with whey protein concentrate (WPC) on the growth performance, histopathological condition of organs, economic efficiency, disease resistance to intraperitoneal inoculation of Aeromonas hydrophila, and the immune response of Oreochromis niloticus. The toxicity of WPC was tested by measuring the activity of caspase 3 as an indicator of cellular apoptosis. Oreochromis niloticus fingerlings with average initial weight 18.65 ± 0.05 gm/fish (n = 225) for a 10-week feeding trial. The fish were randomly allocated to five experimental groups, having five replacement percentages of fish meal with WPC: 0%, 13.8%, 27.7%, 41.6%, and 55.5% (WPC0, WPC13.8, WPC27.7, WPC41.6, and WPC55.5); zero percentage represented the control group. The results show that the fish fed WPC had the same growth performance as the WPC0. The total weight of bacterially challenged surviving fish increased linearly and quadratically (p ≤ 0.05) by increasing the replacement percentage of fish meal with WPC. The growth hormone, nitric oxide, IgM, complement 3, and lysozyme activity were seen to increase significantly in WPC27.7, especially after a bacterial challenge. The phagocytic percentage and phagocytic index increased significantly in WPC27.7, WPC41.6, and WPC55.5 groups. Histopathological examination of liver sections was badly affected by high replacement in WPC41.6–55.5. The activity of caspase 3 in the immunohistochemical stained sections of the intestine was increased significantly by increasing the inclusion level of WPC. Economically, the total return of the total surviving fish after the bacterial challenge was increased significantly by fish meal replacement with WPC. It could be concluded that WPC could replace the fish meal in Nile tilapia diets up to 27.7%, with improving the gut health, the total weight of survival fish, and immune status of fish challenged with A. hydrophila. High inclusion levels of WPC are not recommended in fish diets, since they negatively affected the intestinal and liver tissues and increased the level of cellular apoptosis, as indicated by the increased caspase 3 activity. Further researches are recommended to evaluate the effect of fish meal replacement with WPC on the histopathological examination of the kidney and to test the capacity of serum IgM to clot the bacteria used for the challenge.