Previous work suggests that the relationship between the carbon isotope composition of air (δ13Ca) and plant leaf tissue (δ13Cp) can be used to track changes in the carbon isotope composition of paleo-atmospheric CO2. Here we test this assertion in a series of experiments using the model plant Arabidopsis thaliana grown under a range of atmospheric CO2 concentrations relevant to geologic time (380, 760, 1000, 1500, 2000 and 3000 ppm). Nested within these CO2 experiments water availability was controlled (giving two sets of experimental plants; low and high water treatment at each CO2 concentration) to manipulate stomatal opening, a key process governing carbon fixation and isotope discrimination. Results show a highly significant relationship between δ13Ca and δ13Cp under both experimental water treatments. To test the utility of δ13Cp to predict δ13Ca we compare calculated δ13Ca to measured values of δ13Ca. These data show that although there is a significant relationship between calculated and measured δ13Ca, there is disparity between the two values of δ13Ca and a large difference between calculated values under different water treatments even when grown in a common CO2 concentration. These results demonstrate that environmental factors that alter stomatal opening can severely impact on the use and reliability of δ13Cp to predict δ13Ca and as such, results should be interpreted with caution.