Isoprene (C5H8) emissions by terrestrial vegetation vary with temperature and light intensity, and play an important role in biosphere–chemistry–climate interactions. Such interactions were probably substantially modified by Pleistocene climate and CO2 cycles. Central to understanding the nature of these modifications is assessment and analysis of how emissions changed under glacial environmental conditions. Currently, even the net direction of change is difficult to predict because a CO2-depleted atmosphere may have stimulated emissions compensating for the negative impacts of a cooler climate. Here, we address this issue and attempt to determine the direction of change from an experimental standpoint by investigating the interaction between isoprene emissions and plant growth of two known isoprene-emitting herbaceous species (Mucuna pruriens and Arundo donax) grown at glacial (180 ppm) to present (366 ppm) CO2 levels. We found a significant enhancement of isoprene emissions per unit leaf area in M. pruriens under subambient CO2 concentrations relative to ambient controls but not for A. donax. In contrast, canopy emissions remained unaltered for both plant species because enhanced leaf emissions were offset by reductions in biomass and leaf area. Separate growth experiments with M. pruriens revealed that lowering day/night temperatures by 5°C decreased canopy isoprene emissions irrespective of the CO2 level. Incorporation of these results into a simple canopy emissions model highlights their potential to attenuate reductions in the total isoprene flux from forests under glacial conditions predicted by standard models.