Sub-lethal studies of chlorpyrifos, O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate on mosquito fish, Gambusia affinis were carried out in vivo, for 20 days to assess the locomotor behavior in relation to bioaccumulation and interaction with a targeted enzyme, acetylcholinesterase (AChE, EC: 184.108.40.206). Fish exposed to sub-lethal concentration of 60 Ag/L (1/5 of LC50) were under stress, and reduced their locomotor behavior like distance travelled per unit time (m/min) and swimming speed (cm/sec) with respect to the length of exposure. The alteration in locomotor behavior of fish may be due to an accumulation of acetylcholine (ACh), a neurotransmitter at synaptic junctions, due to the inhibition of AChE enzyme activity (40 to 55%) in brain and also bioaccumulation of the toxicant in different parts of fish. The bioaccumulation values indicated that the accumulation of chlorpyrifos was maximum in viscera followed by head and body. The average bioconcentration values are 0.109, 0.009 and 0.004 Ag/g for viscera, head and body with depuration rates of 2.24, 1.69 and 0.39 ng/h respectively. It is evident from the results that the sub-lethal concentration [1/5 of LC50; equivalent to Lowest Observed Effect Concentration (LOEC)] of chlorpyrifos can able to alter the locomotor behavior of G. affinis in relation to the length of exposure. The findings revealed that the locomotor activity of test organism could be considered as a suitable marker to evaluate the affect of toxicant even at LOEC levels.