Insect–microbial symbiont relationships are widespread in nature and often involve lateral gene transfer. Although the evolutionary processes that allow insects to adapt to complex environments remain largely unknown, it is clear that symbiotic relationships have essential roles in these processes. Here, gut microbes-mediated regulation of Propylea japonica insecticide tolerance is found through modulation of a horizontally transferred gene (P. japonica Domain unknow funcation 1, PjDUF1) expression. However, this gene regulates the host capacity for dinotefuran tolerance by affecting the oxidative phosphorylation rate. This is confirmed by the RNAi-Mediated Silencing of PjDUF1. Importantly, evidence is found that PjDUF1 is donated from Acenitobacter via horizontal gene transfer (HGT). The findings provide the first experimental evidence that HGT events are important for pesticide tolerance in a prominent natural enemy species. Further study of the evolutionary origins of key natural enemy tolerance genes will shed additional light on how insects have developed resistance to adverse environments, suggesting strategies for protecting insect species that provide critical ecosystem services.