Soil excess ammonium (NH4 +) has serious negative effects on crop growth and yield formation. Most studies of NH4 + stress have addressed only the effects of free NH4 +, failing to recognize changes in nitrogen (N) assimilation products. Hydroponic experiments were conducted using 5 mM NH4 + or nitrate (NO3 −) forms of N in two wheat cultivars that differed in NH4 + tolerance (NH4 +-sensitive AK58 and NH4 +-tolerant XM25). To evaluate the effects of NH4 + assimilation products on plant growth, 1 μM L-methionine sulfoximine (MSO, an inhibitor of glutamine synthetase) and 1 mM glutamate (a primary N assimilation product) were added to the N source solutions. NH4 + treatments signiﬁcantly reduced plant biomass in both cultivars, but to a greater extent in AK58. These inhibition effects were alleviated by MSO and strengthened by the application of glutamate. The free NH4 + concentration was increased under NH4 + conditions and was enhanced by the application of MSO. Amino acids levels were significantly greater in both cultivars; however, XM25 showed a lower glutamate concentration and a lower ratio of (glutamate + glutamine) to (aspartic acid + asparagine) than AK58. Under NH4 + conditions, levels of tricarboxylic acid (TCA) cycle intermediates and adenosine triphosphate were decreased due to inhibited pyruvate kinase activity; this inhibition was enhanced by the application of glutamate, but relieved by MSO. These results show that, aside from the large amounts of free NH4 + in wheat tissues, as a primary N assimilation product, the over-accumulated glutamate is also involved in interrupting TCA cycle metabolism under NH4 + stress, leading to reduced plant growth.