While nitrogen (N) derived from ammonium would be energetically less expensive than nitrate-derived N, the use of ammonium-based fertilizer is limited by the potential for toxicity symptoms. Nevertheless, previous studies have shown that exposure to elevated CO
2
favors ammonium assimilation in plants. However, little is known about the impact of different forms of N fertilizer on stomatal opening and their consequent effects on CO
2
and H
2
O diffusion in wheat plants exposed to ambient and elevated CO
2
. In this article, we have examined the response of the photosynthetic machinery of durum wheat (
Triticum durum
, var. Amilcar) grown with different types of N fertilizer (NO
3
−
, NH
4
+
, and NH
4
NO
3
) at 400 versus 700 ppm of CO
2
. Alongside gas exchange and photochemical parameters, the expression of genes involved in CO
2
(
PIP1.1
and
PIP2.3
) and H
2
O (
TIP1
) diffusion as well as key C and N primary metabolism enzymes and metabolites were studied. Our results show that at 400 ppm CO
2
, wheat plants fertilized with ammonium as the N source had stress symptoms and a strong reduction in stomatal conductance, which negatively affected photosynthetic rates. The higher levels of
PIP1.1
and
PIP2.3
expression in ammonium-fertilized plants at 400 ppm CO
2
might reflect the need to overcome limitations to the CO
2
supply to chloroplasts due to restrictions in stomatal conductance. This stomatal limitation might be associated with a strategy to reduce ammonium transport toward leaves. On the other hand, ammonium-fertilized plants at elevated CO
2
did not show stress symptoms, and no differences were detected in stomatal opening or water use efficiency (WUE). Moreover, similar gene expression of the aquaporins
TIP1
,
PIP1.1,
and
PIP2.3
in ammonium-fertilized plants grown at 700 ppm compared to nitrate and ammonium nitrate plants would suggest that an adjustment in CO
2
and H
2
O diffusion is not required. Therefore, in the absence of a stress context triggered by elevated CO
2
, ammonium- and ammonium nitrate-fertilized plants were able to increase their photosynthetic rates, which were translated eventually into higher leaf protein content.