We carried out a simulation experiment in a laboratory in order to study the effects of hydrostatic pressure from 0.1 MPa to 1.0 MPa on nitrogen cycles at the water-sediment interface of a freshwater reservoir. The results show that high hydrostatic pressure facilitates the release of nitrogen at the interface and ammonification, but has little influence on denitrification. And high hydrostatic pressure will also significantly increase the activity of dehydrogenases and proteases in the sediment, and has little influence on ureases and nitrate reductase activity. The pressure caused a large accumulation of such pollutants as ammoniacal nitrogen and nitrate nitrogen at the interface, seriously worsening the overlying water quality. As PLFA and PCR-DGGE analysis results show, different hydrostatic pressures will lead to remarkable differences in the microbial community’s structure and heredity. Within the range 0.1 MPa to 1.0 MPa, the microbial community structure is more diverse under high hydrostatic pressure than under normal pressure.