Glyphosate-resistant (GR) soybean [Glycine max (L.) Merr.] expressing an insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) gene has revolutionized weed control in soybean production. The soybean nitrogen fixing symbiont, Bradyrhizobium japonicum, possesses a glyphosate-sensitive enzyme and upon exposure to glyphosate accumulates shikimic acid and hydroxybenzoic acids such as protocatechuic acid (PCA), accompanied with B. japonicum growth inhibition and death at high concentrations. In a series of greenhouse and field experiments, glyphosate inhibited nodulation and nodule leghemoglobin content of GR soybean. Glyphosate accumulated in nodules of field-grown GR soybean, but its effect on nitrogenase activity of GR soybean was inconsistent in field studies. In greenhouse studies, nitrogenase activity of GR soybean following glyphosate application was transiently inhibited especially in early growth stages, with the greatest inhibition occurring under moisture stress. Studies using bacteroid preparations showed that the level of glyphosate inhibition of bacteroid nitrogenase activity was related to in vitro glyphosate sensitivity of the B. japonicum strains. These studies indicate the potential for reduced nitrogen fixation in the GR soybean system; however, yield reductions due to this reduced N2 fixation in early stages of growth have not been demonstrated.