Tobacco (Nicotiana tabacum L.) expressing a modified Escherichia coli gdhA gene encoding NADPH‐dependent glutamate dehydrogenase (GDH) was resistant to the herbicide glufosinate. Differential plant sensitivity to glufosinate may result from changes in foliar absorption, translocation, plant metabolism, or metabolism around the blocked pathway(s). The objectives here were to determine if foliar absorption, translocation, or metabolism of glufosinate or metabolism around the blocked pathway(s) contribute to the resistance to glufosinate in gdhA‐transformed tobacco and to characterize changes in the metabolic profile in response to glufosinate as a result of transformation with gdhA. Foliar uptake and translocation of glufosinate was largely independent of the gdhA transgene or its expression. Thus, gdhA does not confer resistance to the transformed plants by altering the uptake or movement of glufosinate within tobacco plants. Using direct‐injection electrospray ionization and quadrupole time‐of‐flight mass spectrometry, the metabolic profiles of the two tobacco genotypes were different in response to glufosinate treatment, as determined by principal component analysis. The metabolic perturbation induced by glufosinate was lower in gdhA line 9 than gusA line 1, as evidenced by the reduced number of altered peaks recorded in leaves. Thus, gdhA‐transformed tobacco plants with low and high expression of GDH activity showed more stability of metabolism following the application of glufosinate than control plants. The predicted identity of ions suggested metabolic perturbations were lessened by gdhA in nitrogen assimilation, photorespiration, amino acid metabolism, nucleic acid metabolism, and cell signaling. Thus, use of the modified E. coli gdhA gene in transgenic plants can provide an additional mechanism for resistance to glufosinate.