Subacute studies of profenofos on mosquito fish, Gambusia affinis, were carried out for 20 days to assess the locomotor behavior and structural integrity of gill in relation to bioaccumulation and targeted enzyme acetylcholinesterase (AChE; EC 3.1.1.7). The sublethal concentration of 0.13 mg/L (1/5 of LC50) altered locomotor behavior such as distance traveled and swimming speed in exposed fish. This could be due to inhibition in the activity of acetylcholinesterase and deformities in the primary and secondary lamella of gill. The bioaccumulation values indicated that the accumulation of profenofos was highest in viscera followed by head and body. The average bioconcentration factor values are 254.83, 6.18, and 2.52 microg/g for viscera, head, and body. The findings revealed that profenofos is highly toxic even at sublethal concentrations to the mosquito fish, Gambusia affinis.