The stimulation of plant growth by elevated CO2 concentration has been widely observed. Such fertilization, and associated carbon storage, could dampen future increases in atmospheric CO2 levels and associated climate warming1. However, the CO2 fertilization of plant biomass may be sensitive to nitrogen supply 2-4. Herein we show that in the latest decade of a long-term perennial grassland experiment, low ambient soil nitrogen availability constrained the positive response of plant biomass to elevated CO 2, a result not seen in the first years (1998-2000) of the study. From 2001 to 2010, elevated CO2 stimulated plant biomass half as much under ambient as under enriched nitrogen supply, an effect mirrored over this period by more positive effects of elevated CO2 on soil nitrogen supply (net nitrogen mineralization) and plant nitrogen status under enriched than ambient nitrogen supply. The results did not strongly support either the progressive nitrogen limitation hypothesis, or the alternative hypothesis of priming of soil nitrogen release by elevated CO2. As nitrogen limitation to productivity is widespread, persistent nitrogen constraints on terrestrial responses to rising CO2 are probably pervasive. Further incorporation of such interactions into Earth system models is recommended to better predict future CO2 fertilization effects and impacts on the global carbon cycle.