The widespread application of isoproturon (IPU) can cause serious pollution to the environment and threaten ecological functions. In this study, the IPU bacterial N-demethylase gene pdmAB was transferred and expressed in the chloroplast of soybean (Glycine max L. 'Zhonghuang13'). The transgenic soybeans exhibited significant tolerance to IPU and demethylated IPU to a less phytotoxic metabolite 3-(4-isopropylphenyl)-1-methylurea (MDIPU) in vivo. The transgenic soybeans removed 98% and 84% IPU from water and soil within 5 and 14 days, respectively, while accumulating less IPU in plant tissues compared with the wild-type (WT). Under IPU stress, transgenic soybeans showed a higher symbiotic nitrogen fixation performance (with higher total nodule biomass and nitrogenase activity) and a more stable rhizosphere bacterial community than the WT. This study developed a transgenic (TS) soybean capable of efficiently removing IPU from its growing environment and recovering a high-symbiotic nitrogen fixation capacity under IPU stress, and provides new insights into the interactions between rhizosphere microorganisms and TS legumes under herbicide stress.