Reconstruction of the carbon isotope composition of atmospheric CO2 is critical to the understanding of long-term global carbon cycling. We have suggested that the δ13C value of land plant carbon (δ13Cp) preserved in the geologic record should reflect the δ13CO2 at the time during which the plants grew (δ13Ca), based on a meta-analysis of modern plant data. Here we present the results of laboratory experiments designed to quantify the relationship between plant tissue δ13C and δ13CO2 values under varying environmental conditions, including differential pCO2 ranging from 1 to 3 times today's levels. As predicted, plants grown under elevated pCO2 showed increased average biomass compared to controls grown at the same temperature. Across a very large range in δ13Ca (≈24‰) and pCO2 (≈740 ppmv) we observed a consistent correlation between δ13Ca and δ13Cp (p < 0.001). We show an average isotopic depletion of −25.4‰ for aboveground tissue and −23.2‰ for belowground tissue of Raphanussativus L. relative to the composition of the atmosphere under which it formed. For aboveground and belowground tissue, grown at both ∼23°C and ∼29°C, correlation was strong and significant (r2 ≥ 0.98 and p < 0.001); variation in pCO2 level had little or no effect on this relationship. These results validate our initial conclusion that in the absence of environmental stress, plant δ13C primarily reflects atmospheric δ13CO2 linearly across pCO2 levels; the demonstrated excellent correlation in δ13Ca and δ13Cp suggests a high level of predictive power across varying environmental conditions.