Among plants grown under enriched atmospheric CO2, root:shoot balance (RSB) theory predicts a proportionately greater allocation of assimilate to roots than among ambient-grown plants. Conversely, defoliation, which decreases the plant's capacity to assimilate carbon, is predicted to increase allocation to shoot. We tested these RSB predictions, and whether responses to CO2 enrichment were modified by defoliation, using Heterotheca subaxillaris, an annual plant native to south-eastern USA. Plants were grown under near-ambient (400 μmol mol−1) and enriched (700 μmol mol−1) levels of atmospheric CO2. Defoliation consisted of the weekly removal of 25% of each new fully expanded, but not previously defoliated, leaf from either rosette or bolted plants. In addition to dry mass measurements of leaves, stems, and roots, Kjeldahl N, protein, starch and soluble sugars were analysed in these plant components to test the hypothesis that changes in C:N uptake ratio drive shifts in root:shoot ratio. Young, rapidly growing CO2-enriched plants conformed to the predictions of RSB, with higher root:shoot ratio than ambient-grown plants (P < 0.02), whereas older, slower growing plants did not show a CO2 effect on root:shoot ratio. Defoliation resulted in smaller plants, among which both root and shoot biomass were reduced, irrespective of CO2 treatment (P < 0.03). However, H. subaxillaris plants were able to compensate for leaf area removal through flexible shoot allocation to more leaves vs. stem (P < 0.01). Increased carbon availability through CO2 enrichment did not enhance the response to defoliation, apparently because of complete growth compensation for defoliation, even under ambient conditions. CO2-enriched plants had higher rates of photosynthesis (P < 0.0001), but this did not translate into increased final biomass accumulation. On the other hand, earlier and more abundant yield of flower biomass was an important consequence of growth under CO2 enrichment.