To improve understanding of spatial, seasonal, and inter-annual variations in nitrate sources and in-stream processes in the Illinois River system, nitrate concentrations and isotopic compositions were measured in 445 water samples collected over a four-year period (2004–2008) from the Upper Illinois River Basin (UIRB). Samples included surface water in the river and major tributaries, effluent samples from Chicago’s largest wastewater treatment plant (WTP), and representative groundwater from shallow wells in agricultural land. Two principal nitrate endmember sources within the UIRB had distinctive isotopic compositions: WTP effluent with δ¹⁵N = 8.6 ± 1.7‰ and δ¹⁸O = 0.8 ± 1.4‰ and agricultural groundwater with δ¹⁵N-NO₃ = 3.4 ± 0.6‰ and δ¹⁸O = 3.7 ± 0.5‰ (when minimally affected by nitrate reduction). Isotopic data indicated that the large pulse of nitrate exported from the river basin during the spring was mostly derived from agricultural land drainage, while nitrate from large WTP effluent point sources was predominant in the upper reaches of the river near Chicago. During low base-flow conditions in late-summer and fall, the agricultural nitrate source was greatly diminished and the headwater WTP source was predominant in the river basin export. Our results indicated biogeochemical nitrate reduction and isotopic fractionation occurred within the river network, affecting both agricultural and urban sources during surface-water transport. In addition, diminished agricultural nitrate export was attributable to preferential discharge of biogeochemically reduced groundwater during low base flow. Isotopic indicators of spatial and seasonal variations in the relative importance of different nitrate sources, and their relative susceptibility to natural attenuation, might be useful for guiding monitoring and management practices to reduce nitrate export from complex watersheds with mixed land uses.