Macrophytes influence nitrogen (N) removal from wetlands. However, the specific plant traits responsible for this effect and the related microbial mechanisms remain largely unknown, especially root traits. In a mesocosm experiment, we determined the rhizosphere effect (RE) on microbial N removal processes by incubating rhizosphere and bulk soils collected from 11 macrophyte species. In addition, we examined root traits (involved in chemistry and morphology), along with examining the diversity, compositions, and abundance of bacterial communities involved in denitrification (nirS and nirK) and anammox (hzsB). Across the 11 macrophyte species, the positive RE on denitrification ranged from 66% to 412%, with an average of 194.72%. RE on denitrification was significantly and positively correlated with the recruitment of nir-type denitrifiers in the rhizosphere. We found that higher specific root length (SRL) root promoted the stronger RE, by increasing the abundance of nir-type denitrifiers and further enhancing N removal. Net N removal from water in the wetlands increased with a higher positive RE on nir-type denitrifiers. In addition, SRL significantly influenced the compositions of denitrifiers in the rhizosphere soil. We further found that the enrichment of Azospira, Bradyrhizobium, Sinorhizobium, Rhodopseudomonas, Alcaligenaceae, Bradyrhizobiaceae, and Pleomorphomonas improved the denitrification rate. These findings highlight the potential of root morphology in regulating plant–microbe interactions, thereby improving water purification.