Abstract: In order to explore the effects of Bacillus thuringiensis(Bt) on the midgut tissue and hemolymph enzyme activities of Dendrolimus kikuchii larvae, herein the 3rd instar larvae, which were incubated from the cocoons of D. kikuchii collected in Anning City and reared in the lab., were used as test worms. Furthermore, the virulence of Bt preparation was determined based on Probit regression method, and then the larvae were infected with lethal concentration 50(LC₅₀) at the best detection time point. Moreover, after infection, the midgut tissue lesions were examined under a microscope at different times, and the activities of phenol oxidase(PO), carboxylesterase(CarE) and acetylcholinesterase(AChE) in hemolymph were measured. The results showed that 48 h is the best detection time for the virulence determination of Bt preparations, with the LC₅₀ of 13.024 IU/μL; the typical midgut lesion sobserved were as follows: elongated and deformed columnar cells, the significantly increased number of goblet cells, the markedly enlarged cell cavity, and the vanished periphagus membrane, etc. The enzymatic activities of CarE, AChE and PO in the hemolymph almost were significantly increased, compared to the larvae without treatment with Bt(the blank control), that of AChE was the most obvious. The results would provide a theoretical basis for the biological control of D. kikuchii.