Abstract With the advent of glyphosate [N-(phosphonomethyl)glycine] tolerant crops, soils have now been receiving repeated applications of the herbicide for over 10 years in the Midwestern USA. There is evidence that long-term use of glyphosate can cause micronutrient deficiency but little is known about plant potassium (K) uptake interactions with glyphosate. The repeated use of glyphosate may create a selection pressure in soil microbial communities that could affect soil K dynamics and ultimately K availability for crops. Therefore, the objectives of this study were to characterize the effect of foliar glyphosate applied to GR (glyphosate resistant) soybeans on: (1) rhizosphere microbial community profiles using ester linked fatty acid methyl ester (EL-FAME) biomarkers, (2) exchangeable, non-exchangeable, and microbial K in the rhizosphere soil, and (3) concentrations of soybean leaf K. A greenhouse study was conducted in a 2 × 2 × 3 factorial design with two soil treatments (with or without long-term field applications of glyphosate), two plant treatments (presence and absence of soybean plants), and three rates of glyphosate treatments (0×, 1× at 0.87, and 2× at 1.74 kg ae ha −1 , the recommended field rate). After each glyphosate application, rhizosphere soils were sampled and analyzed for microbial community structure using ester linked fatty acid methyl ester biomarkers (EL-FAME), and exchangeable, plant tissue and microbial biomass K. Glyphosate application caused a significant decrease in the total microbial biomass in soybean rhizosphere soil that had no previous exposure to glyphosate, at 7 days after glyphosate application. However, no significant changes were observed in the overall microbial community structure. In conclusion, the glyphosate application lowered the total microbial biomass in the GR soybean rhizosphere soil that had no previous exposure to glyphosate, at 7 days after glyphosate application; caused no changes in the microbial community structure; and did not reduce the plant available K (soil exchangeable or plant tissue K).