Ammonium (NH4 +) serves as a nitrogen source, but its elevated levels can hinder plant growth and production. Excess NH4 + with α-ketoglutarate is assimilated into glutamate, a precursor of proline and glutathione (GSH). This study aimed to investigate the effects of excessive NH4 + on the regulation of proline and GSH synthesis. Detached leaves from oilseed rape (Brassica napus L.) were fed with 0, 50, 100, 500, and 1000 mM NH4Cl for 16 h. As the NH4 + concentrations increased, the leaves exhibited progressive wilting and yellowing. Furthermore, total carotenoid and chlorophyll concentrations declined in response to all NH4 + treatments, with the lowest levels observed in 1000 mM NH4 + treatment. Hydrogen peroxide (H2O2) concentration showed a minor increase at low NH4 + concentration (50 and 100 mM) treatments but a significant increase at high NH4 + (500 and 1000 mM), which was consistent with the localization of H2O2. Amino acid concentrations increased with increasing in NH4 + concentration, while the protein concentration displayed the opposite trend. Proline and cysteine concentrations exhibited a gradual increase in response to increasing NH4 + concentrations. However, GSH concentrations rose only in the 50 mM NH4 + treatment and decreased in the 500 and 1000 mM NH4 + tre...