Previous greenhouse studies have demonstrated that photosynthesis in some cultivars of first- (GR1) and second-generation (GR2) glyphosate-resistant soybean was reduced by glyphosate. The reduction in photosynthesis that resulted from glyphosate might affect nutrient uptake and lead to lower plant biomass production and ultimately reduced grain yield. Therefore, a field study was conducted to determine if glyphosate-induced damage to soybean (Glycine max L. Merr. cv. Asgrow AG3539) plants observed under controlled greenhouse conditions might occur in the field environment. The present study evaluated photosynthetic rate, nutrient accumulation, nodulation, and biomass production of GR2 soybean receiving different rates of glyphosate (0, 800, 1200, 2400 g a.e. ha–1) applied at V2, V4, and V6 growth stages. In general, plant damage observed in the field study was similar to that in previous greenhouse studies. Increasing glyphosate rates and applications at later growth stages decreased nutrient accumulation, nodulation, leaf area, and shoot biomass production. Thus, to reduce potential undesirable effects of glyphosate on plant growth, application of the lowest glyphosate rate for weed-control efficacy at early growth stages (V2 to V4) is suggested as an advantageous practice within current weed control in GR soybean for optimal crop productivity.