The feasibility of using a constructed wetland for treatment of nitrate-contaminated groundwater resulting from the land application of biosolids was investigated for a site in the southeastern United States. Biosolids degradation led to the release of ammonia, which upon oxidation resulted in nitrate concentrations in the upper aquifer in the range of 65–400 mg N/L. A laboratory-scale system was constructed in support of a pilot-scale project to investigate the effect of temperature, hydraulic retention time (HRT) and nitrate and carbon loading on denitrification using soil and groundwater from the biosolids application site. The maximum specific reduction rates (MSRR), measured in batch assays conducted with an open to the atmosphere reactor at four initial nitrate concentrations from 70 to 400 mg N/L, showed that the nitrate reduction rate was not affected by the initial nitrate concentration. The MSRR values at 22 °C for nitrate and nitrite were 1.2 ± 0.2 and 0.7 ± 0.1 mg N/mg VSS COD-day, respectively. MSRR values were also measured at 5, 10, 15 and 22 °C and the temperature coefficient for nitrate reduction was estimated at 1.13. Based on the performance of laboratory-scale continuous-flow reactors and model simulations, wetland performance can be maintained at high nitrogen removal efficiency (>90%) with an HRT of 3 days or higher and at temperature values as low as 5 °C, as long as there is sufficient biodegradable carbon available to achieve complete denitrification. The results of this study show that based on the climate in the southeastern United States, a constructed wetland can be used for the treatment of nitrate-contaminated groundwater to low, acceptable nitrate levels.