The effects of 2,4-D (1 or 10 mg/L) on acetylcholinesterase (AChE) and metabolic parameters were evaluated in piava (Leporinus obtusidens) after 96 h. AChE activity was significantly reduced in the brain at a concentration of 10 mg/L and in the muscle at both concentrations tested. Muscle glycogen and lactate were significantly reduced for both 2,4-D concentrations but no significant change was observed in liver glycogen. Muscle protein levels were enhanced after exposure at 10 mg/L, but no significant changes were observed in muscle and liver glucose. Liver lactate and protein were significantly reduced after exposure to this herbicide. 2,4-D exposure produced a decrease in blood glucose at both concentrations and enhanced lactate levels at 10mg/L. Plasma protein increased at both concentrations tested. In conclusion, the results obtained indicate that 2,4-D affects brain and muscle AChE activity and some blood and tissue metabolic parameters of L. obtusidens. The stress generated by 2,4-D is the probable cause of alterations observed and measured parameters can be used to monitor 2,4-D fish toxicity.