Leaf‐level measurements have shown that mesophyll conductance (gm) can vary rapidly in response to CO2 and other environmental factors, but similar studies at the canopy‐scale are missing. Here, we report the effect of short‐term variation of CO2 concentration on canopy‐scale gm and other CO2 exchange parameters of sunflower (Helianthus annuus L.) stands in the presence and absence of abscisic acid (ABA) in their nutrient solution. gm was estimated from gas exchange and on‐line carbon isotope discrimination (Δobs) in a 13CO2/12CO2 gas exchange mesocosm. The isotopic contribution of (photo)respiration to stand‐scale Δobs was determined with the experimental approach of Tcherkez et al. Without ABA, short‐term exposures to different CO2 concentrations (Ca 100 to 900 µmol mol−1) had little effect on canopy‐scale gm. But, addition of ABA strongly altered the CO2‐response: gm was high (approx. 0.5 mol CO2 m−2 s−1) at Ca < 200 µmol mol−1 and decreased to <0.1 mol CO2 m−2 s−1 at Ca >400 µmol mol−1. In the absence of ABA, the contribution of (photo)respiration to stand‐scale Δobs was high at low Ca (7.2‰) and decreased to <2‰ at Ca > 400 µmol mol−1. Treatment with ABA halved this effect at all Ca.