Most soybean (Glycine max L. Merr.) cultivars grown in the U.S. are Roundup Ready®, genetically modified for resistance to the herbicide glyphosate. The objectives of this study were to examine the impacts of glyphosate applications on symbiotic N 2 fixation in glyphosate-resistant (GR) soybean and to evaluate the responses of rhizobial isolates and nifH gene abundance in the rhizosphere soil to glyphosate. In two repeated greenhouse experiments, GR soybean received foliar applications of glyphosate once or twice during the study period; the untreated GR and near-isogenic conventional cultivar served as controls. Plants were harvested twice, two days after each glyphosate application. In addition to plant growth parameters, the nitrogenase activity of root nodules and the abundance of the nifH gene in the rhizosphere were determined using the acetylene reduction assay and quantitative PCR, respectively. Glyphosate-treated GR soybean had lower chlorophyll content, root mass, nodule mass, total plant N, and nitrogenase activity than the untreated conventional cultivar, especially for the second harvest. Without glyphosate application, few differences were observed between the two cultivars. Glyphosate inhibited the growth of rhizobia isolated from root nodules; however, the nifH gene abundance in the rhizosphere was not different among the treatments. Glyphosate appeared to exert stress to the GR soybean cultivar used in this study. Further research is needed to verify the greenhouse experiment findings and possible yield effects under field conditions.