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Herbicides are the most commonly used means of controlling weeds. Recently, there has been growing concern over the potential impacts of global climate change, specifically, increasing temperatures and elevated carbon dioxide (CO2) concentrations, on the sensitivity of weeds to herbicides. Here, glyphosate response of both Conyza canadensis and Chenopodium album was evaluated under different environmental conditions. Reduced glyphosate sensitivity was observed in both species in response to increased temperature, elevated CO2 level, and the combination of both factors. Increased temperature had greater effect on plant survival than elevated CO2 level. In combination, high temperature and elevated CO2 level resulted in loss of apical dominance and rapid necrosis in glyphosate-treated plants. To investigate the mechanistic basis of reduced glyphosate sensitivity, translocation was examined using C-14-glyphosate. In plants that were subjected to high temperatures and elevated CO2 level, glyphosate was more rapidly translocated out of the treated leaf to shoot meristems and roots than in plants grown under control conditions. These results suggest that altered glyphosate translocation and tissue-specific sequestration may be the basis of reduced plant sensitivity. Therefore, overreliance on glyphosate for weed control under changing climatic conditions may result in more weed control failures.