Previous research has demonstrated that glyphosate can affect nitrogen fixation or nitrogen assimilation in soybean. This 2-year field study investigated the effects of glyphosate application of 1.12 and 3.36 kg of ae ha(-1) on nitrogen metabolism and seed composition in glyphosate-resistant (GR) soybean. There was no effect of glyphosate application on nitrogen fixation as measured by acetylene reduction assay, soybean yield, or seed nitrogen content. However, there were significant effects of glyphosate application on nitrogen assimilation, as measured by in vivo nitrate reductase activity (NRA) in leaves, roots, and nodules, especially at high rate. Transiently lower leaf nitrogen or (15)N natural abundance in high glyphosate application soybean supports the inhibition of NRA. With the higher glyphosate application level protein was significantly higher (10.3%) in treated soybean compared to untreated soybean. Inversely, total oil and linolenic acid were lowest at the high glyphosate application rate, but oleic acid was greatest (22%) in treated soybean. These results suggest that nitrate assimilation in GR soybean was more affected than nitrogen fixation by glyphosate application and that glyphosate application may alter nitrogen and carbon metabolism.