In vitro, high nitrate (NO3 (-)) concentrations significantly inhibit N2O reductase activity. However, little information is available on the in situ temporal effects of excessive N fertilization on soil N2O reductase activity and the regulation of the N2O/(N-2 + N2O) product ratio in agricultural soil. This study examined the monthly in situ dynamics of NO3 (-) concentration, N2O reductase activity, and N2O/(N-2 + N2O) product ratio for 2 years in loamy soil that had received either continuous N fertilizer at 400 kg N ha(-1) year(-1) for 15 years (N400) or no N fertilizers (CK). N2O reductase activity was significantly lower under the N400 treatment than under the CK and correlated negatively with soil NO3 (-) concentration. The decrease in N2O reductase activity resulted in the N2O/(N-2 + N2O) product ratio increasing. These results demonstrate that excessive N fertilization has the potential to increase N2O emissions by reducing N2O reductase activity in soils. These results highlight the need for N2O mitigation options to embrace the reduction of soil NO3 (-) concentrations.