Operational conditions are often manipulated to improve the nitrogen removal performance of wastewater treatment, yet the impacts of operational conditions on microbial communities were still not well understood. There is a pressing need to understand the microbial mechanisms that link operation manipulation and nitrogen removal performance. In this study, high-throughput analysis of 16S rDNA and quantitative polymerase chain reaction of functional genes were used to identify the microbial response to operational manipulations. The results showed that alteration of operational parameters could change the bacterial communities at the genera level and denitrification guild gradually dominated in the activated sludge bacterial communities. Heterotrophic Hyphomicrobium and Chromatiaceae drove the kinetic of dominant genera and denitrification guild. Carbon source supplement was the most efficient strategy for improving nitrogen removal, and greatly increased the abundance of denitrifiers and denitrification genes. However, carbon source supplement inhibited expression activities of denitrification genes, as well as the proliferation of autotrophic denitrifiers, and it was supposed to be unfavorable in terms of cost over the long term. The result should bring new inspiration for improving the effect of WWTP performance through the manipulation of operational parameters.