Wheat is a major staple food crop worldwide contributing approximately 20% of total protein consumed by mankind. The nitrogen and protein concentration of wheat crop and grain often decline as a result of exposure of the crop to elevated CO2 (EC). The changes in nitrogen (N) assimilation, root system architecture, and nitric oxide (NO)-mediated N signaling and expression of genes involved in N assimilation and high affinity nitrate uptake were examined in response to different nitrate levels and EC in wheat. Activity of enzyme nitrate reductase (NRA) was downregulated under EC both in leaf and root tissues. Plants grown under EC displayed enhanced production of NO and more so when nitrate supply was high. Based on exogenous supply of NO, inhibitors of NO production, and NO scavenger, regulatory role of NO on EC mediated changes in root morphology and NRA was revealed. The enhanced NO production under EC and high N levels negatively regulated the transcript abundance of NR and high affinity nitrate transporters (HATS).