Acid rain has degraded environmental health since the Industrial Revolution. Legislative efforts have 19 successfully reduced deposition rates, but recovery of affected ecosystems in the Mississippi River Basin 20 (MRB) remains to be assessed. Combining analysis of temporal trends of indicative chemical species in 21 streams and rivers with a Bayesian statistical model, we found strong evidence of reduced effect by acid 22 rain on water chemistry; however, the effect by agricultural activities and climate change are intensifying. 23 pH increased in the Eastern MRB, the historically more heavily affected region, and SO4 loading 24 decreased everywhere, suggesting recovery. Widespread fertilizer use, however, has likely accelerated 25 carbonate weathering and water acidification. As a result, water became more acidic in western sites and 26 annual divalent cation (DIV) load increased at all sites, showing statistically significant trends. Extended 27 dry summers under climate change have likely contributed to SO4 and DIV export via shale weathering in 28 the basin, when the groundwater table drops. We also found evidence that, while not a significant 29 contributor yet, ever increasing atmospheric CO2 levels will likely add to cation export from the MRB in 30 the future. Using long-term data over a large spatial scale, this study represents a comprehensive 31 assessment of the recovery of water chemistry in river and stream ecosystems from acid rain in the 32 Mississippi Basin, taking into consideration the entangled effects of agricultural activities, acid mine 33 drainage, and extended droughts and elevated atmospheric CO2 concentration under anthropogenic 34 climate change.