Increasing groundwater and soil salinity is a threat to the land and water resources in arid regions. Global warming will likely increase salinity of dryland river systems. In order to characterize salt loading into the semi-arid portion of the Rio Grande in south New Mexico and west Texas, we sampled seasonally (2009-2011) the river, agricultural drains, and saline groundwater. In addition to major element chemistry, these samples were analyzed for sulfur and oxygen isotope compositions (δ34S and δ18O) of dissolved SO4 and in some cases for nitrogen and oxygen isotope compositions (delta;15N and δ18O) of dissolved NO3. Uranium isotopes (234U/238U activity ratio) were also measured for selected samples. The natural inflow of basinal brines/groundwater (δ34S of + 8 to + 11‰) in the semi-arid Rio Grande study area was minor in the investigated seasons and could not be detected by the δ34S mass balance. However, we did find localized increases of δ34S (+ 2 to + 5‰) in the Rio Grande that were attributable to salt loads from the intersections of agricultural drains with the water table of a natural salt flat and associated evaporative brine (δ34S of + 12‰) in the shallow subsurface. In the areas, with higher water use for land irrigation, the δ34S of the river and drain water was relatively consistent (from ~ 0 to + 2‰) compared to the δ18O (from ~+ 2 to + 6‰). Most likely, this resulted from application of S-rich fertilizers (e.g., ammonium sulfates, elemental S, sulfuric acid) with low δ34S (- 2 to + 4‰) and high δ18O (+ 9 to + 16‰). Additionally, we observed considerably lower δ18O (SO4) in the Rio Grande and agricultural drains (> 7‰) compared to geologic and anthropogenic SO4 sources (+ 9 to + 16‰), which likely resulted from microbial recycling of SO4 in soil of the irrigated land related to assimilatory sulfate reduction. Shallow recharge to the Rio Grande was also inferred from the lower 234U/238U activity ratios (1.62 to 1.88) compared to deeper groundwater (2.54 to 2.64) and the distinctive delta;15N and δ18O values of nitrates (+ 5 to + 25‰ and - 5 to + 15‰, respectively) typical for septic effluents that are undergoing denitrification. Agricultural practices during flood irrigation intensify evaporation of the Rio Grande surface water and considerably increase water salinity. This process is also important in the evolution of water chemistry toward a Na-SO4-Cl-rich composition and precipitation of secondary calcite in soil profiles.