Recent studies have found insignificant or decreasing trends in time-series dissolved organic carbon (DOC) datasets, questioning the assumption that long-term DOC concentrations in surface waters are increasing in response to anthropogenic forcing, including climate change, land use, and atmospheric acid deposition. We used the weighted regressions on time, discharge, and season (WRTDS) model to estimate annual flow-normalized concentrations and fluxes to determine if changes in DOC quantity and quality signal anthropogenic forcing at 10 locations in the Mississippi River Basin. Despite increases in agriculture and urban development throughout the basin, net increases in DOC concentration and flux were significant at only 3 of 10 sites from 1997 to 2013 and ranged between −3.5% to +18% and −0.1 to 19%, respectively. Positive shifts in DOC quality, characterized by increasing specific ultraviolet absorbance at 254 nm, ranged between +8% and +45%, but only occurred at one of the sites with significant DOC quantity increases. Basinwide reductions in atmospheric sulfate deposition did not result in large increases in DOC either, likely because of the high buffering capacity of the soil. Hydroclimatic factors including annual discharge, precipitation, and temperature did not significantly change during the 17-year timespan of this study, which contrasts with results from previous studies showing significant increases in precipitation and discharge over a century time scale. Our study also contrasts with those from smaller catchments, which have shown stronger DOC responses to climate, land use, and acidic deposition. This temporal and spatial analysis indicated that there was a potential change in DOC sources in the Mississippi River Basin between 1997 and 2013. However, the overall magnitude of DOC trends was not large, and the pattern in quantity and quality increases for the 10 study sites was not consistent throughout the basin.