Abstract: To clarify the acetylcholinesterase (AChE) changes among different developmental stages of Tribolium castaneum (Herbst), the toxicological and biochemical characterizations of AChE in 10-d larvae, 20-d larvae, pupae and adults were investigated, respectively with the microplate reader method. The results showed that there were significant differences of protein content and the AChE activity and specific activity among the four developmental stages of T. castaneum. The protein contents of AChE were 10-d larvae < 20-d larvae < pupae < adults, while the AChE activity and specific activity were adults < pupae < 10-d larvae < 20-d larvae. The kinetic analysis of AChE revealed that the apparent Michaelis-Menten constants (Km) by magnitude were as follows: pupae < adults <10-d larvae < 20-d larvae. And no significant difference appeared between the Km values of pupae and adults. In addition, the maximum velocity (Vmax) in larvae was significant higher than those in pupae and adults. In vitro inhibition studies of larvae AChE indicated that the I50 values of 20-d larvae were higher than those of 10-d larvae when treated with eserine, malaoxon and carbaryl, respectively, suggesting that AChE in 20-d larvae was less sensitive to the inhibitors.