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Bacillus thuringiensis cotton seeds are treated with insecticides for protection against pathogenic organism’s infestation. The use of insecticides as seed treatments entails both economic benefits and potential risks like phytotoxicity. The present study was carried out to evaluate the biochemical effect of majorly used neonicotinoid seed treatment insecticide imidacloprid (trade name Gaucho 70 WS) at recommended concentration (3.2g/Kg seeds) on the growth of seedlings (length, biomass) of three Bt cotton hybrids (RCH-134, JKCH-1947, NCEH-6R), by analyzing their defense related parameters on third and eighth day after sowing of treated seeds under controlled conditions. It was observed that imidacloprid increased the activity of defense enzyme peroxidase significantly w.r.t controls (at p<0.01) on 8th day after germination and non significantly on 3rd day in both RCH-134 and JKCH-1947 hybrids. Superoxide dismutase activity showed significant increase (at p<0.01) from 1.71±0.53 ΔE/min/mg protein to 6.72±1.23 ΔE/min/mg protein in NCEH-6R plumules and at p<0.05 in JKCH-1947 plumules from 0.71±0.13 ΔE/min/mg protein to 4.95±0.52 ΔE/min/mg protein. A non significant increase was found in Phenylalanine ammonia lyase activity in all three hybrid seedlings . The levels of Bacillus thuringiensis protein increased significantly (at p<0.01) in RCH-134 and NCEH-6R seedlings on 3rd day after germination and (at p<0.05) on 8th day after germination. Total phenols were also increased significantly (at p<0.01) with respect to control seedlings in all three Bt cotton hybrid seedlings on both days of observation. Length and biomass of seedlings showed no significant and variable increase. The results suggested that the seed treatment with imidacloprid enhanced the defense system of the seedlings and might help to increase the cotton crop quality and productivity.