Canada thistle was grown under field conditions in 2000 and 2003 at ambient and elevated (∼ 350 μmol mol−1 above ambient) carbon dioxide [CO2] to assess how rising [CO2] alters growth, biomass allocation, and efficacy of the postemergent herbicide glyphosate. By the time of glyphosate application, approximately 2 mo after emergence, elevated CO2 had resulted in significant increases in both root and shoot biomass. However, the relative positive effect of [CO2] was much larger for root, relative to shoot growth, during this period (2.5- to 3.3-fold vs. 1.2- to 1.4-fold, respectively) with a subsequent increase in root to shoot ratio. Glyphosate was applied at 2.24 kg ae ha−1 in 2000 and 2003. Subjective classification of leaf damage in shoots after spraying indicated no significant difference in the extent of necrosis in aboveground tissue as a function of CO2 concentration. After a 6-wk regrowth period, significant reductions in shoot and root biomass relative to unsprayed plots were observed under ambient [CO2]. However, the decrease in the ratio of sprayed to unsprayed biomass was significantly less at elevated relative to ambient [CO2] conditions for roots in both years, and no difference in shoot biomass was observed between sprayed and unsprayed plots for Canada thistle grown at elevated [CO2] in either year. The observed reduction in glyphosate efficacy at the enriched [CO2] treatment did not appear to be associated with differential herbicide uptake, suggesting that tolerance was simply a dilution effect, related to the large stimulation of root relative to shoot biomass at elevated [CO2]. Overall, the study indicates that carbon dioxide–induced increases in root biomass could make Canada thistle and other perennial weeds that reproduce asexually from belowground organs harder to control in a higher [CO2] world.