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Questions Intensification of livestock production has led to the increasing input of agrochemicals, mostly herbicides, in natural and semi-natural grasslands. Glyphosate application shifts vegetation composition of grasslands of the Flooding Pampa region. Consequently, we asked two questions: (1) does the application of glyphosate affect the size, composition and diversity of the seed bank of these grasslands; and (2) to what extent does seed bank determine the vegetation composition of grassland communities affected by glyphosate application? Location Temperate-humid grassland of Flooding Pampa region, Buenos Aires, Argentina. Method We studied paddocks dominated by native grassland on a commercial livestock farm. Glyphosate is usually applied in late summer to promote winter annual grasses in native grasslands. We chose three paddocks that had never been exposed to any herbicide and three paddocks that had been treated with glyphosate in late summer during the previous 5 years. We extracted soil cores to evaluate seed bank composition from 2007 to 2009 and recorded basal cover of above-ground vegetation from 2008 to 2010 from the upper and the lower position of each paddock. Results Glyphosate treatment caused a dramatic shift in seed bank composition, so that seed densities of cool-season annual grasses increased while cool and warm-season perennial grasses, sedges, legumes and dicotyledonous herbs decreased. Richness and diversity of the seed bank were lower and dominance was higher under the glyphosate treatment. Similarity between the seed bank and floristic composition was higher in glyphosate-treated paddocks in the upper position. Strong correspondences between the seed densities of cool-season annual grasses and species composition of glyphosate-treated paddocks and between the seed densities of cool- and warm-season perennial grasses and legumes with the species composition of untreated paddocks were found. Conclusions Because the seed bank changes in response to glyphosate treatment involved the local extinction of several native perennial species, community restoration of these grasslands would depend mainly on propagule dispersal from adjacent areas. Our results raise awareness of the risks of widespread herbicide application for biodiversity conservation in the last semi-natural habitats in the Pampas grasslands.