Aims We aimed to develop a biological agent that regulates the microbial community structure of the poplar rhizosphere and alleviates the effects of continuous poplar cropping. Methods and Results Poplar rhizosphere soils were treated with or without Paenibacillus polymyxa CP-S316 microbial fermentation medium. Real-time polymerase chain reaction was performed to measure bacteria and fungi in both groups, and microbial communities were analysed by metabarcoding. In fungi, the operational taxonomic units, abundance-based coverage estimator and Chao index of the CP-S316-treated group were significantly lower than those in the control check (CK) group. In bacteria, the proportions of Bacillus in the CP-S316 and CK groups were 5 center dot 20 and 2 center dot 38%, respectively, whereas those of Rhizoctonia were 2 center dot 20 and 5 center dot 82% respectively. The diameter at breast height, culturable bacteria and total bacteria of poplars treated with CP-S316 exceeded those in the CK group. Conclusions Our data confirmed that CP-S316 could improve the microbial community structure of poplar rhizosphere and promote the growth of poplars. Significance and Impact of the Study Research aimed at alleviating continuous cropping obstacles and promoting poplar growth via biocontrol agents is uncommon. We analysed the community structures of bacteria and fungi in rhizosphere soil to illustrate the use of CP-S316 for poplar cropping for improving plant health in the continuous cropping of poplar trees.