An investigation to determine whether stomatal acclimation to [CO2] occurred in C3/C4 grassland plants grown across a range of [CO2] (200-550 μmol mol-1) in the field was carried out. Acclimation was assessed by measuring the response of stomatal conductance (gs) to a range of intercellular CO2 (a gs-Ci curve) at each growth [CO2] in the third and fourth growing seasons of the treatment. The gs-Ci response curves for Solanum dimidiatum (C3 perennial forb) differed significantly across [CO2] treatments, suggesting that stomatal acclimation had occurred. Evidence of non-linear stomatal acclimation to [CO2] in this species was also found as maximum gs (gsmax; gs measured at the lowest Ci) increased with decreasing growth [CO2] only below 400 μmol mol-1. The substantial increase in gs at sub-ambient [CO2] for S. dimidiatum was weakly correlated with the maximum velocity of carboxylation (Vcmax; r2 = 0.27) and was not associated with CO2 saturated photosynthesis (Amax). The response of gs to Ci did not vary with growth [CO2] in Bromus japonicus (C3 annual grass) or Bothriochloa ischaemum (C4 perennial grass), suggesting that stomatal acclimation had not occurred in these species. Stomatal density, which increased with rising [CO2] in both C3 species, was not correlated with gs. Larger stomatal size at subambient [CO2], however, may be associated with stomatal acclimation in S. dimidiatum. Incorporating stomatal acclimation into modelling studies could improve the ability to predict changes in ecosystem water fluxes and water availability with rising CO2 and to understand their magnitudes relative to the past.