We compared biological functions of two acetylcholinesterase genes (TcAce1 and TcAce2) in Tribolium castaneum, a globally distributed major pest of stored grain products and an emerging model organism, by using RNA interference. Although both genes expressed at all developmental stages and mainly in the brain, the transcript level of TcAce1 was 1.2- to 8.7-fold higher than that of TcAce2, depending on developmental stages. Silencing TcAce1 in 20-day larvae led to 100% mortality within two weeks after eclosion and increased larval susceptibilities to anticholinesterase insecticides. In contrast, silencing TcAce2 did not show insect mortality and significantly affect insecticide susceptibility, but delayed insect development and reduced female egg-laying and egg hatching. These results demonstrate for the first time that TcAce1 plays a major role in cholinergic functions and is the target of anticholinesterase insecticides, whereas TcAce2 plays an important, non-cholinergic role in female reproduction, embryo development, and growth of offspring.