Aldehyde oxidases (AOXs) are a group of metabolic enzymes that play critical roles in the degradation of xenobiotics and chemicals. However, the physiological function of this enzyme in insects remains poorly understood. In this study, three TcAOX genes (TcAOX1, TcAOX2, TcAOX3) were identified and characterized from Tribolium castaneum genome. Spatiotemporal expression profiling showed that TcAOX1 expression was most highly expressed at the early pupal stage and was predominantly expressed in the antennae of adults, indicating that TcAOX1 was involved in the degradation of chemical signals; TcAOX2 expression was most highly expressed at the late pupal stage and was mainly expressed in the fat body, epidermis of larvae and adults, respectively; and TcAOX3 expression was in all stages and was primarily expressed in the head of adults. Moreover, the transcripts of TcAOX2 and TcAOX3 were significantly induced after exposure to plant oil, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to this plant toxicant, suggesting that these two genes are associated with plant toxicant detoxification. Intriguingly, knockdown of the TcAOX1 led to reductions in female egg-laying but unchanged the hatchability and the development of genital organs, suggesting that this gene may mediate fecundity by effecting the inactivation of chemical signals in T. castaneum. Overall, these results shed new light on the function of AOX genes in insects, and could facilitate the development of research on pest control management.