The effects of diflubenzuron (DFB), an insecticide to control ectoparasites in fish farms, on muscle acetylcholinesterase (AChE), detoxifying and antioxidant enzymes, hematological and physiological parameters, and liver histopathology were evaluated in Prochilodus lineatus after 6, 24 and 96 h of exposure to 25 mg L(-1) of DFB. The insecticide caused a reduction in the number of erythrocytes and hemoglobin content after 96 h exposure, probably due to hemolysis. Hyperglycemic response indicated energy mobilization, and may have contributed to the increase in osmolarity after 96 h exposure to DFB. The induction of glutathione-S-transferase (GST) and catalase activities in liver pointed to the activation of xenobiotic metabolic pathways and antioxidant defenses. The decrease in muscle AChE at all experimental times showed that DFB is an AChE inhibitor. In addition, DFB induced hepatic alterations that might impair normal liver functions. These results show that DFB can cause health disorders in fish and further studies are required to better define its safe use in aquaculture.