Abstract: In China, there are an increasing number of ponds receiving sewage effluents with high concentrations of nitrogen (N) and dissolved oxygen (DO) for agricultural purposes. Therefore, it is worthwhile to understand the essential N transformation processes as well as the characteristics of N-cycling bacteria driving these processes under the condition of high DO concentrations. In order to evaluate the diel vertical variation of denitrifying bacteria in the water column of the ponds during the process of accumulation and storage of sewage, an experiment was carried out to monitor the changes of denitrifying genes abundance and chemical and physical characteristics at the different depths of sewage water reserved in a pond at Jiangsu Academy of Agricultural Sciences, Nanjing, China. The water samples were collected from a sampling site where had the most stable hydrodynamic condition and least disturbance due to the discharge of sewage water during irrigation season. Water samples were collected from the different water depths of 10-30, 90-110 and 150-170 cm, periodically (5 consecutive time points, 12:00 pm, 0:00 am, 4:00 am, 08:00 am and 12:00 pm at the next day), within 24 hours during 15th -16th August 2013. The collected samples were then analyzed with the abundances of denitrifying genes (nirK, nirK, and nosZ) using real-time polymerase chain reaction (PCR), and water parameters including pH, water temperature, illuminance and the concentrations of DO, NO3--N, NH4+-N and PO43--P . The correlations between the changes of nirK, nirK, and nosZ genes abundance and water parameters were statistically analyzed. The results showed that the strong and persistent stratification formed along vertical profile of sewage water with regards to all above monitored physical and chemical parameters. The relatively higher pH, water temperature, illuminance, and the concentrations of DO and NO3--N were observed in the surface water layers (10-30 cm), decreasing with the increase of water depths. On the contrast, the higher concentrations of NH4+-N and PO43--P were observed in the bottom water, which means that they were increased with the increase of water depths. Due to high photosynthetic activity of microscopic algae in the pond, the concentrations of DO concentration increased during the daylight and decreased during night. Diurnal variation of pH was positively (P<0.001) correlated with DO concentration in the vertical profile of the water column in the pond, with pH in the ponds also followed the diurnal cycle of sunlight intensity. The abundances of nirK, nirK, and nosZ genes were strongly affected by the diurnal distribution patterns of pH and DO concentrations along vertical profile of water，with significant higher abundance during the daytime while lower abundance during the night at all the different water depths. Heterogeneous distribution of denitrifying bacteria abundances along the vertical profile of sewage water column were also demonstrated, with the least abundances in the bottom water (150-170 cm) and the highest in the surface water column (10-30 cm). Although the nirK and nirS are functionally equivalent, denitrifying bacteria harboring either nitrite reductase seem to be likely not under the same community assembly rules. The abundance of nirS gene was always greater than that of nirK gene in all water samples, suggesting that the sewage water in this study was more suitable for the growth of nirS-type denitrifying bacteria. This speculation was consistent with previously identified habitat preferences of nirS- and nirK-gene bearing organisms. The abundances of nirK, nirS, and nosZ genes were significantly correlated with DO concentration and pH (P<0.01). These results demonstrated that the diel vertical variability of the water physical and chemical parameters, especially DO concentrations, in the oxygen-enriched sewage water could affect the diel vertical distribution patterns of nirK, nirS, and nosZ genes abundances. This may suggest that a large number of aerobic denitrifying bacteria could have extensively developed in sewage water under the condition of high DO concentrations during the process of conservation in the pond. These aerobic denitrifying bacteria can have an important impact on the nitrogen transformation processes in the vertical profile of sewage water column.