Growth room studies were conducted to determine the physiological basis of reduced glyphosate efficacy under low soil nitrogen using velvetleaf, common ragweed, and common lambsquarters as model species. Glyphosate dose–response experiments of weeds grown under low (1.5 mM) and high (15 mM) soil N were conducted. Velvetleaf and common lambsquarters grown under low N required 169 g ae ha−1 glyphosate for a significant reduction in biomass, but only 84 g ae ha−1 were required when grown under high N. However, when common ragweed was grown under low or high soil N there was no significant difference in response to glyphosate at all doses tested. The reduced glyphosate efficacy on velvetleaf and common lambsquarters under low N was primarily due to decreased herbicide translocation to the meristem. It appears that low N may decrease the net assimilation of carbon in plants, resulting in a decrease in the net export of sugars and hence glyphosate from mature leaves. Understanding the relationship between soil N and herbicide efficacy may help explain observed weed control failures with glyphosate and may contribute to our knowledge of the occurrence of weed patchiness in fields. This is the first report illustrating a physiological basis for decreased glyphosate efficacy under low soil N in selected weed species.