Elevated carbon dioxide (e-CO₂) levels from ambient (a-CO₂) enhance plant biomass production and yield. However, this response is highly dependent on the availability and possibly the form of nitrogen (N) supply to plants. This study aimed to investigate changes in C and N metabolism of wheat (Triticum aestivum L.) in response to e-CO₂ and N source. e-CO₂ enhanced net CO₂ assimilation but at highly variable rates depending on the form of N supply. Under e-CO₂, net CO₂ assimilation rate was in the order NO₃^– > NH₄NO₃ > NH₄⁺ > urea. Plants supplied with ammonium and nitrate (i.e. NH₄NO₃) performed better in terms of biomass production and expressed a higher biomass enhancement ratio by e-CO₂ than plants receiving sole applications of NO₃^–, NH₄⁺ or urea. Supply of NH₄NO₃ also resulted in lower intercellular CO₂, higher photoassimilate translocation to roots and lower accumulation of free amino acids than other N forms, indicating a better exploitation of the e-CO₂ environment. Our results conclude that major physiological pathways of photosynthesis and protein and carbohydrate metabolism are differentially influenced by e-CO₂ depending on the source of N supply. A balanced supply of NO₃^– and NH₄⁺ to plant roots is the key to harnessing e-CO₂ while minimising its adverse effects on quality of the produce.