The main objective of this work was to quantify and update the U.S. Midwest agricultural state of Iowa’s contribution of nitrate-nitrogen to the Mississippi River stream network against the backdrop of the ongoing problem of Gulf of Mexico hypoxia. To achieve this objective, we used stream nitrate and discharge data collected from 1999 until 2016 at 23 Iowa stream sites near watershed outlets, along with publicly-available data for sites downstream of Iowa on the Missouri and Mississippi Rivers. Our analysis shows that Iowa contributes between 11 and 52% of the long-term nitrate load to the Mississippi-Atchafalaya Basin, 20 to 63% to the Upper Mississippi River Basin, and 20 to 89% to the Missouri River Basin, with averages of 29, 45 and 55% respectively. Since 1999, nitrate loads in the Iowa-inclusive basins have increased and these increases do not appear to be driven by changes in discharge and cropping intensity unique to Iowa. The 5-year running annual average of Iowa nitrate loading has been above the 2003 level for ten consecutive years, implying that Gulf hypoxic areal goals, also based on a 5-year running annual average, will be very difficult to achieve if nitrate retention cannot be improved in Iowa. An opportunity exists for land managers, policy makers and conservationists to manifest a positive effect on water quality by targeting and implementing nitrate reducing-practices in areas like Iowa while avoiding areas that are less likely to affect Gulf of Mexico hypoxia.