Although legumes showed a clearly superior yield response to elevated atmospheric pCO2 compared to nonlegumes in a variety of field experiments, the extent to which this is due to symbiotic N2 fixation per se has yet to be determined. Thus, effectively and ineffectively nodulating lucerne (Medicago sativa L.) plants with a very similar genetic background were grown in competition with each other on fertile soil in the Swiss FACE experiment in order to monitor their CO2 response. Under elevated atmospheric pCO2, effectively nodulating lucerne, thus capable of symbiotically fixing N2, strongly increased the harvestable biomass and the N yield, independent of N fertilization. In contrast, the harvestable biomass and N yield of ineffectively nodulating plants were affected negatively by elevated atmospheric pCO2 when N fertilization was low. Large amounts of N fertilizer enabled the plants to respond more favourably to elevated atmospheric pCO2, although not as strongly as effectively nodulating plants. The CO2-induced increase in N yield of the effectively nodulating plants was attributed solely to an increase in symbiotic N2 fixation of 50–175%, depending on the N fertilization treatment. N yield derived from the uptake of mineral N from the soil was, however, not affected by elevated pCO2. This result demonstrates that, in fertile soil and under temperate climatic conditions, symbiotic N2 fixation per se is responsible for the considerably greater amount of above-ground biomass and the higher N yield under elevated atmospheric pCO2. This supports the assumption that symbiotic N2 fixation plays a key role in maintaining the C/N balance in terrestrial ecosystems in a CO2-rich world.