Nitrate contamination of water bodies is a serious concern in regions with intensive agriculture. Previous studies, which only considered surface water and groundwater but not the regolith, do not provide a comprehensive accounting of nitrate sources and their proportional contributions in the Critical Zone, which extends from the top of the vegetation canopy down to the bottom of the fresh groundwater. In this study, dual nitrate isotopes, hydrochemical compositions and a Bayesian isotope mixing model were used to investigate nitrate sources, processes and individual source contributions across surface water, regolith and groundwater at the Red Soil Critical Zone Observatory of China. Denitrification was found to be weak in the Critical Zone. Dilution processes were important in eliminating nitrate from streamflow and groundwater. More than 90% of the nitrate inventory was stored at depths from 1 m to the bedrock surface in the upland regolith. Nitrate with light nitrogen (¹⁴N) isotopes was preferentially adsorbed by the variable charge soils with the help of weak electrostatic attraction. The contribution of soil organic N (SON) to the nitrate in the upland catchment streamflow was found to be highest with a mean of 56%, followed by manure and sewage (MS, 26%), NH₄⁺ in chemical fertilizers (CF, 17%). For the paddy subcatchment streamflow, the contribution of SON (68%) was highest, followed by CF (19%) and MS (13%). In contrast, for the groundwater, the contributions of CF and SON were 60% and 36%, respectively. For the regolith, the contributions of CF and SON were 72% and 25%, respectively. Thus the most important sources of nitrate were SON for the streamflow and CF for the groundwater and regolith. These findings show that the different partitioning and isotopic fractionation within the regolith strongly affect the sources of nitrate reaching groundwater, compared to the sources reaching surface water.