The role of floating macrophytes on modulating the microbial nitrogen removal is not well understood. In this study, the cultivation of Eichhornia crassipes in eutrophic water may affect the nitrogen (N) fate by modulating the denitrifying bacteria diversity and abundance. The gaseous N losses via denitrification were estimated by 15N stable isotope tracing and the diversity and abundance of denitrifying genes (nirS, nirK, and nosZ) were investigated by molecular tools. The denaturing gradient gel electrophoresis (DGGE) profiles showed that the diversity of denitrifying genes in the treatments with E. crassipes was significantly higher than that in the treatment without E. crassipes. The real‐time PCR (qPCR) results showed the trend of denitrifier abundance in the entire system was in the order of N‐ER (nitrate with just root of E. crassipes) and A‐ER (ammonia with just root of E. crassipes) > N‐R (nitrate with E. crassipes) and A‐R (ammonia with E. crassipes) > N‐W (nitrate without plant) and A‐W (ammonia without plant). The gaseous 15N losses via denitrification were significantly and positively related to the abundance of nirK, nirS, and nosZ genes. The results indicated that cultivation of E. crassipes in eutrophic water could increase the diversity and abundance of denitrifying bacteria, resulting in more N being removed as gases via denitrification.