Previous research demonstrated that plant nutrient assimilation was reduced by glyphosate (Gly). A 2 year field experiment investigated the effects of Gly at drift rate (12.5% of commercial use rate) on Fe concentrations in leaves and seeds of Gly-sensitive (GS) soybean, and a greenhouse experiment evaluated Gly effects on Fe assimilation using root in vivo ferric reductase activity (FRA) in two GS and one Gly-resistant (GR) soybean cultivars. Field studies showed that Gly drift rates resulted in a significant decrease in the Fe concentration in seeds and leaves compared to the nontreated plants. In greenhouse studies, leaf Fe and FRA were inhibited in GS cultivars Hutcheson and DP 5110 and the GR cultivar AG 4604RR and leaf Fe was positively correlated with root FRA (p < 0.0001). These results indicate that Gly can interfere with Fe assimilation in both GS and GR soybean. Understanding the implication of Gly on Fe nutrition in soybean seed would help soybean agronomists and breeders seeking to improve seed mineral nutrition qualities.