Chironomids commonly dominate macroinvertebrate assemblages in aquatic habitats and these non-biting midges are known for their ability to tolerate contaminants. Studies regarding the interplay between gut microbiota and host detoxification ability is currently a point of interest. Cytochrome P450s (P450s) are critical metabolic enzymes in which a subset is involved in xenobiotic detoxification. In this study, we first conducted an integrated global investigation of P450s based on the whole genomic sequence of Propsilocerus akamusi and retrieved a series of 64 P450 genes which were further classified into 4 clans and 25 families on the basis of phylogenetic relationships. With assistance of RNA-Seq and RT-qPCR validation, the expression profile of screened PaP450s in guts was compared between chlorpyrifos-challenged larvae with deficient gut microbiota (GD) and those with a conventional gut community (CV). An increasing prevalence of chlorpyrifos from sublethal to lethal dosages induced a greater mortality rate of individuals coupled with remarkable downregulation of 14 P450s in GD larval guts when compared to CV ones. Moreover, it turned out that the decreased level of PaCYP3998B1 and PaCYP3987D1 might imply impaired host endogenous detoxification capability potentiated by gut dysbiosis, reflected by a remarkably severe mortality in GD larvae treated with lethal chlorpyrifos. Collectively, our study unveiled candidate P450 genes that might be mediated by gut symbionts in chlorpyrifos-challenged P. akamusi larvae, possibly facilitating further understanding of the detoxified mechanism that chironomids might employ to alleviate poisonousness.