Nutrient reduction strategies in the Mississippi and Atchafalaya River Basin (MARB) have been implemented to attenuate drinking water concerns and hypoxia in the northern Gulf of Mexico. Of all nutrients, nitrate has been identified as the principal cause of Gulf hypoxia, with loads coming disproportionately from the Upper Mississippi River Basin and the Corn Belt region. Identifying long-term changes of riverine nitrate would provide valuable information to evaluate the performance of reduction strategies. The objective of this study was to estimate the flow-normalized (FN) nitrate-N concentration and yield trends for the 2000-2020 period across the MARB at monitoring sites with adequate data. A harmonization and in-depth screening of paired nitrate-N and streamflow datasets resulted in a robust water quality monitoring network of 217 sites. Trends magnitude and likelihood were computed using the Weighted Regression on Time, Discharge, and Season (WRTDS) coupled to a bootstrap test, and trends results were correlated with basin features and initial values of concentrations and yields. The impact of streamflow long-term variations on trends was separated from all other factors through stationary and non-stationary flow normalizations. Results indicated that 59.4 % of the 217 sites had likely decreasing concentration trends, while 27.7 % likely increased, and the remaining 12.9 % had no likely change detected. Reductions in riverine FN nitrate-N were predominant at watersheds dominated by cultivated cropland areas having relatively high FN concentrations and yields in 2000 followed by likely downward trends. For the vast majority of sites, the non-streamflow component was more dominant, but the streamflow component was nonetheless influential.