The concentration of carbon dioxide in Earth’s atmosphere may double by the end of the 21st century. The response of higher plants to a carbon dioxide doubling often includes a decline in their nitrogen status, but the reasons for this decline have been uncertain. We used five independent methods with wheat and Arabidopsis to show that atmospheric carbon dioxide enrichment inhibited the assimilation of nitrate into organic nitrogen compounds. This inhibition may be largely responsible for carbon dioxide acclimation, the decrease in photosynthesis and growth of plants conducting C3 carbon fixation after long exposures (days to years) to carbon dioxide enrichment. These results suggest that the relative availability of soil ammonium and nitrate to most plants will become increasingly important in determining their productivity as well as their quality as food.