Prior to discharging to the ocean, large rivers constantly receive inputs of dissolved organic carbon (DOC) from tributaries or fringing floodplains and lose DOC via continuous in situ processing along distances that span thousands of kilometers. Current concepts predicting longitudinal changes in DOC mainly focus on in situ processing or exchange with fringing floodplain wetlands, while effects of heterogeneous watershed characteristics are generally ignored. We analyzed results from a 17-year time-series of DOC measurements made at seven sites and three expeditions along the entire Mississippi River main channel with DOC measurements made every 17 km. The results show a clear downstream decrease in DOC concentrations that was consistent throughout the entire study period. Downstream DOC decreases were primarily (∼63–71%) a result of constant dilutions by low-DOC tributary water controlled by watershed wetland distribution, while in situ processing played a secondary role. We estimate that from 1780 to 1980 wetland loss due to land-use alterations caused a ca. 58% decrease in in DOC concentrations in the tributaries of the Mississippi River. DOC reductions caused by watershed wetland loss likely impacted the capacity for the river to effectively remove nitrogen via denitrification, which can further exacerbate coastal hypoxia. These findings highlight the importance of watershed wetlands in regulating DOC longitudinally along the headland to ocean continuum of major rivers.