Hexaflumuron (HEX) insecticide is widely used in agriculture practices to fight crop insects. The toxicological effect of HEX on Nile tilapia (Oreochromis niloticus) was investigated in this study. Two hundred and forty fish (35.50 ± 1.45 g) were divided into six groups in four replicates (40 fish/group; 10 fish/replicate) and were exposed to six distinct HEX concentrations (0, 2, 4, 6, 8, and 10 mg L−1) for 96-h. The 96-h lethal concentration 50 (96-h LC50) of HEX was calculated to be 7.19 mg L−1. The fish exhibited reduced surface and middle swimming, aggressiveness, and tail-spreading behaviors with increasing bottom swimming and resting patterns after HEX exposure. HEX exposure resulted in body bleeding and f in rot. The erythrogram (red blood cell count, hemoglobin, and packed cell volume %) was significantly reduced with increased mean corpuscular volume by HEX exposure. HEX exposure decreased the white blood cells (WBCs) and dif ferential WBC counts. Acute HEX exposure raised 8-hydroxy-2-deoxyguanosine level while lowering brain acetylcholine esterase activity. HEX exposure caused hepato-renal dysfunction and increased stress-related parameters (glucose and cortisol). Exposure to HEX reduced the immune responses (lysozyme, nitric oxide, immunoglobulin M, and complement 3). A substantial decrease in the antioxidant variables (reduced glutathione content and catalase) with increasing the malo ndialdehyde was noted by HEX exposure. Moreover, histopathological changes resulted from HEX exposure in the gills, liver, kidney, and spleen. These results indicate that HEX exposure induced behavioral changes, hepato-renal dysfunction, and immune-antioxidant disruption, indicating a possible physiological disruption in O. niloticus.