Impact assessment of sluice regulation on water quality is one of the crucial tasks in the present river management. However, research difficulties remain because of insufficient in situ data and numerous influencing factors in aquatic environments. The Huaidian Sluice, the main control sluice of the Shaying River, China, was selected for this study. Three field experimental programs were designed and carried out to analyze spatial and temporal variations in water quality parameters under various sluice regulation conditions and to explore the impacts of regulation mechanisms on water quality. Monitoring data were used to simulate water quality under different scenarios by the water quality analysis simulation program (WASP). Results demonstrate that the influences of sluice regulation on permanganate index (CODMn) and ammonia nitrogen (NH4-N) concentrations (indicators of water quality) were complex and nonlinear and presented different trends of increase or decrease from different regulation modes. Gate openings of different widths and different flow rates affected CODMn and NH4-N concentrations differently. Monitoring results and numerical simulation results indicate that the sluice opening should be small. Flow discharge through the sluice should be greater than 10 m(3) s and less than 60 m(3) s to maintain low CODMn concentrations, and discharge should be low (e.g., 14 m(3) s) to maintain low NH4-N concentrations. This research provides an experimental basis for further research on the construction of water quality models and for the development of reasonable regulations on water quality and quantity.