The root system of five-year-old Populus×euramericana cv.‘Neva’were manually pruned with a sharp spade at distances of six, eight and ten times DBH(diameter at breast height) from both two sides or four sides prior to buds break, to analyze the effects of different root pruning treatments on biological characteristics of the rhizosphere soil. The results indicated that:(1) The root pruning treatment of eight times DBH from two sides significantly increased the concentrations of the available P and organic matter in the rhizosphere soil, and increased total amino acids and total organic acids in the root exudates in contrast to the control. At the same time, the microbial populations and enzymes activities in the rhizosphere soil were also enhanced, showing 20.61%, 18.87%, 36.92% and 22.97% increases in the activities of urease, alkaline phosphatase, polyphenol oxidase and invertase enzymes compared to the control, respectively. However, the root pruning treatment of eight times DBH from two sides obviously decreased the available N concentration and pH value in the rhizosphere soil.(2) In comparison with the root pruning treatment of eight times DBH from two sides, the root pruning treatments of eight times DBH from four sides, ten times DBH from two sides, as well as ten times DBH from four sides had less effect on inproving physio-chemical and biological characteristics of poplar rhizosphere soil, which were still better than the control. In addition, the root pruning treatments of six times DBH from two sides as well as from four sides had the least effect on the physio-chemical and biological characteristics in the rhizosphere soil among all the treatments. In conclusion, the root pruning was beneficial to the ecological environment of rhizosphere soil at the eight times DBH distance along both inter-row sides.