Background: Poplar (Populus) is a genus of globally important plantation trees used widely in industrial and agricultural production. However, poplar is easily damaged by Micromelalopha troglodyta and Hyphantria cunea, resulting in a decline in poplar quality. Due to their strong insect resistance, Bt toxin-encoded Cry genes have been widely adopted in poplar breeding; however, potential adverse effects of Cry1Ah1-modified poplars on the ecological environment have raised concerns. Results: In this study, we comprehensively analyzed the structural and functional composition of the rhizosphere microbiome in field-grown transgenic Bt poplar. Conclusions: Our analysis of soil chemistry patterns revealed that soil alkaline nitrogen, soil available phosphorus, and microbial biomass nitrogen and phosphorus levels were improved, whereas microbial biomass carbon declined in Cry1Ah1-modified poplar rhizosphere samples. We applied metagenomic sequencing of Non-Transgenic (NT) and Cry1Ah1-modified poplar rhizosphere samples collected from a natural field; the predominant taxa included Proteobacteria, Acidobacteria, and Actinobacteria. We also identified microbial functional traits involved in membrane transport, amino acid metabolism, carbohydrate metabolism, and replication and repair in NT and Cry1Ah1-modified poplars. Together, these results demonstrate that the NT and Cry1Ah1-modified poplar rhizosphere microbiomes had similar diversity and structure. These differences in relative abundance were observed in a few genera but did not affect the primary genera or soil.