Denitrification is an important pathway for nitrogen removal from aquatic systems and this could benefit water quality. However, little is known about the denitrifier community composition and key steps of denitrification in the freshwater environments, and whether different bacteria have a role in multiple processes of denitrification reduction. In this study, quantitative PCR, quantitative RT-PCR, clone library and 454 pyrosequencing were used together to investigate the bacterial and denitrifier community in a subtropical deep reservoir during the strongly stratified period. Our results indicated that the narG gene recorded the highest abundance among the denitrifying genes (2.76 × 10⁹ copies L-1 for DNA and 4.19 × 10⁸ copies L^-1 for RNA), and the lowest value was nosZ gene (7.56× 10⁵ copies L^-1 for DNA and undetected for RNA). The RNA: DNA ratios indicated that narG gene was the most active denitrifying gene in the oxygen minimum zone of Dongzhen Reservoir. Further, α-, β- and γ- Proteobacteria were the overwhelmingly dominant classes of denitrifier communities. Each functional gene had its own dominant groups which were different at the genus level: the narG gene was dominated by Albidiferax, while nirS gene was dominated by Dechloromonas. The main OTU of nirK gene was Rhodopseudomonas palustris, but for norB and nosZ genes, they were Bacillus and Bradyrhizobium, respectively. These results contribute to the understanding of linkages between denitrifier community, function and how they work together to complete the denitrification process. Studies on denitrifier community and activity may be useful in managing stratified reservoirs for the ecosystem services and aiding in constructing nitrogen budgets.