Host specificity is observed in gut symbionts of diverse animal lineages. But how hosts maintain symbionts while rejecting their close relatives remains elusive. We use eusocial bees and their codiversified gut bacteria to understand host regulation driving symbiotic specificity. The cross-inoculation of bumblebee Gilliamella induced higher prostaglandin in the honeybee gut, promoting a pronounced host response through immune deficiency (IMD) and Toll pathways. Gene silencing and vitamin C treatments indicate that reactive oxygen species (ROS), not antimicrobial peptides, acts as the effector in inhibiting the non-native strain. Quantitative PCR and RNAi further reveal a regulatory function of the IMD and Toll pathways, in which Relish and dorsal-1 may regulate Dual Oxidase ( Duox ) for ROS production. Therefore, the honeybee maintains symbiotic specificity by creating a hostile gut environment to exotic bacteria, through differential regulation of its immune system, reflecting a co-opting of existing machinery evolved to combat pathogens.