We have previously demonstrated that recurrent application of glyphosate causes dramatic shift in the vegetation structure of the native grasslands of Flooding Pampa. As these structural changes might alter functional processes such as primary production, carbon, nitrogen and phosphorus cycling, this study aims to evaluate functional changes associated with the application of glyphosate in these temperate grasslands. We measured aboveground net primary production (ANPP) during two consecutive years, and the concentration of organic carbon, nitrogen and phosphorus in the soil during the following six years after primary production measurements ended in glyphosate treated and non- treated (control) paddocks of a commercial livestock far. We related the vegetation data, basal cover, species richness and diversity, obtained in a previous study conducted in the same paddocks of the livestock farm, with ANPP data obtained in this one. Late summer applications of glyphosate greatly reduced the biomass contribution of warm-season perennial grasses and legumes and increased the contribution of cool season annual grasses, altering the seasonal pattern of ANPP. As the reduction of the spring and summer productivity could not be compensated by the increase of cool-season productivity, the annual ANPP was lower in the glyphosate-treated paddocks than in control paddocks. Glyphosate applications also decreased soil organic carbon and phosphorus concentration, probably because of the reduction of ANPP, the changes of its seasonal distribution and the shift in the floristic composition of the community, which may modify the amount and quality of the litter. We found a linear positive relationship between basal cover, species richness and species diversity with ANPP, which suggest that the negative effects on ecosystem functioning would be directly related with the changes in vegetation structure caused by glyphosate application.