The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations both in the head (neuronal) and abdomen (non-neuronal) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. The expression levels of another two factors that regulate ACh titer, choline acetyltransferase (AmAChT) and acetylcholinesterase 2 (AmAChE2), were not altered as judged by qPCR and native PAGE, suggesting that the lower ACh titer was mainly regulated by AmAChE1. For precise verification of AmAChE1 as an ACh titer regulator, honey bees were put under brood rearing-suppressed condition to induce AmAChE1 and injected AmAChE1 dsRNA to knock down the gene. The ACh titer of AmAChE1-knocked down honey bees was 1.9 and 2.6 folds higher than that of control bees in head and abdomen, respectively. Taken together, in spite of its extremely low catalytic activity, the overexpression of AmAChE1 is likely to be related with the low level of ACh homeostasis, perhaps via ACh sequestration, under brood rearingsuppressed condition, and likely induce metabolic changes through ACh receptors-related pathways.