This study aimed to determine the effects of root pruning on the physicochemical characteristics and microbial activities of poplar rhizosphere soil. The root systems of 5-year-old poplar (Populus×euramericana cv. 'Neva') trees were manually pruned at 6, 8, or 10 times diameter at breast height (DBH) from the trunk (severe, moderate, and light, respectively) along both inter-row sides. Moderate root pruning significantly increased the concentrations of amino acids, organic acids, and total sugars in the root exudates and decreased the pH of rhizosphere soil. This treatment also increased the contents of available nitrogen, phosphorus, potassium, and total organic carbon as well as high-, medium-, and low-activity organic carbon in rhizosphere soil. Moreover, moderate pruning increased the contents of microbial biomass carbon and nitrogen, and enhanced basal respiration, in addition to decreasing the metabolic quotients in rhizosphere soil by 8.9%, 5.0%, and 11.4% compared with control, light, and severe root pruning treatments, respectively. Moderate pruning increased the growth rates of DBH, tree height, and volume to the highest levels. Furthermore, these indices were not significantly different between the light root pruning and control groups, but varied significantly between severe and moderate root-pruning treatments. Thus, root pruning, depending on the distance from the trunk, significantly influences the physicochemical properties and microbial activities in poplar rhizosphere soil.