Digestive and detoxification enzyme activity and nutrient composition were examined in the body of fourth instar beet armyworms, Spodoptera exigua (Hübner), fed on transgenic Bacillus thuringiensis (Bt) and non-Bt cotton for different time periods. Nutrient composition and specific enzyme activities differed significantly between the S. exigua fed Bt vs. non-Bt cotton. At 1, 6 and 24 h, free fatty acid and glucose levels were significantly lower in S. exigua fed on Bt cotton than those fed on non-Bt cotton. S. exigua fed on Bt cotton had significantly higher trypsin and total superoxide dismutase (T-SOD) activities and significantly lower lipase, carboxylesterase and acetylcholinesterase activities than non-Bt fed worms for all feeding time periods. Differences were also observed among feeding times within each cotton variety group. Significantly lower free fatty acid and total amino acid were observed in S. exigua fed on Bt cotton for 24 h than in those fed for 1 h. Significantly lower activities of lipase and trypsin were detected in S. exigua fed on Bt cotton for 24 h than those for 1 and 4 h. However, carboxylesterase and acetylcholinesterase activities in S. exigua fed on Bt cotton for 24 h were significantly higher than those for 1, 4 and 6 h. The interaction between cotton variety and feeding time significantly affected the activities of lipase, trypsin, acetylcholinesterase and T-SOD enzymes in S. exigua. Measuring the temporal allocation of protection and detoxification enzyme activities in the body of S. exigua in response to B. thuringiensis can provide a meaningful evaluation on the metabolic tolerance of herbivorous insects under the continuous selection pressure of a toxic protein.