Herbicides applied to soils potentially affect soil microbial activity. Quantity and frequency of glyphosate application have escalated with the advent of glyphosate-tolerant crops. The objective of this study was to determine the effect of increasing glyphosate application rate on soil microbial biomass and activity. The soil used was Weswood silt loam. The isopropylamine salt of glyphosate was added at rates of 47, 94, 140, and 234 µg ai g−1 soil based on an assumed 2-mm glyphosate–soil interaction depth. Glyphosate significantly stimulated soil microbial activity as measured by C and N mineralization but did not affect soil microbial biomass. Cumulative C mineralization, as well as mineralization rate, increased with increasing glyphosate rate. Strong linear relationships between mineralized C and N and the amount of C and N added as glyphosate (r 2 = 0.995, 0.996) and slopes approximating one indicated that glyphosate was the direct cause of the enhanced microbial activity. An increase in C mineralization rate occurred the first day following glyphosate addition and continued for 14 d. Glyphosate appeared to be directly and rapidly degraded by microbes, even at high application rates, without adversely affecting microbial activity.