Nitrous oxide (N2O) is widely recognized as one of the most important greenhouse gases, and responsible for stratospheric ozone destruction. A significant fraction of N2O emissions to the atmosphere is from rivers. Reliable catchment-scale estimates of these emissions require both high-resolution field data and suitable models able to capture the main processes controlling nitrogen transformation within surface and subsurface riverine environments. Thus, this investigation tests and validates a recently proposed parsimonious and effective model to predict riverine N2O fluxes with measurements taken along the main stem of the Upper Mississippi River (UMR). The model parameterizes N2O emissions by means of two denitrification Damköhler numbers; one accounting for processes occurring within the hyporheic and benthic zones, and the other one within the water column, as a function of river size. Its performance was assessed with several statistical quantitative indexes such as: Absolute Error (AE), Nash-Sutcliffe efficiency (NSE), percent bias (PBIAS), and ratio of the root mean square error to the standard deviation of measured data (RSR). Comparison of predicted N2O gradients between water and air (ΔN2O) with those quantified from field measurements validates the predictive performance of the model and allow extending previous findings to large river networks including highly regulated rivers with cascade reservoirs and locks. Results show the major role played by the water column processes in contributing to N2O emissions in large rivers. Consequently, N2O productions along the UMR, characterized by regulated flows and large channel size, occur chiefly within this surficial riverine compartment, where the suspended particles may create anoxic microsites, which favor denitrification.