Aim: The variations in locomotory behaviour and enzyme activity, i.e., acetylcholinesterase and general esterases of Spodoptera litura Fabricius larval populations collected from different regions of Punjab, i.e., Amritsar, Kapurthala and Ludhiana were investigated in the laboratory prior exposure to different insecticides. Methodology: The test larvae were exposed to sublethal doses of different insecticides via surface and food treatment. For locomotory behaviour, they were allowed to move on treated surface and their movements were recorded with an automated video tracking system, Ethovision while on other hand food treatment was opted for observing the influence on enzyme activity. Results: The pronounced variations were observed in the locomotor behaviour of S. litura upon surface exposure of different insecticides. The larva travelled more distance with higher speed and larger turn angle on insecticide-treated surfaces than the untreated ones, while on comparison of different larval populations, the Amritsar population larvae travelled less distance with low speed and maximum turnings. The decrease in acetylcholinesterase and general esterase activity was recorded in treated larvae than untreated ones. The influence of emamectin benzoate was recorded more on locomotion and enzyme activity of S. litura as compared to the remaining insecticides and the Amritsar population was least affected. Interpretation: The sublethal doses of insecticides influenced the overall behavioural physiology of S. litura and this biological parameter could be useful in designing the management strategies for S. litura. Key words: Acetylcholinesterase, Esterases, Insecticides, Locomotory behaviour, S. litura