Biogenically released isoprene plays important roles in both tropospheric photochemistry and plant metabolism. We performed a 13CO2-labeling study using proton-transfer-reaction mass spectrometry (PTR-MS) to examine the kinetics of recently assimilated photosynthate into isoprene emitted from poplar (Populus x canescens) when trees are grown and measured at different atmospheric CO2 concentrations. This is the first study to explicitly consider the effects of altered atmospheric CO2 concentration on carbon partitioning to isoprene biosynthesis. We studied changes in the proportion of carbons labeled as a function of time in two mass fragments, M41+, which represents, in part, substrate derived from pyruvate, and M69+, which represents the whole unlabeled isoprene molecule. We observed the trend of slower 13C incorporation into the isoprene carbon derived from pyruvate, consistent with the previously hypothesized origin of chloroplastic pyruvate from cytosolic phosphenolpyruvate (PEP). Trees grown in sub-ambient CO2 (190 ppmv) had rates of isoprene emission and rates of labeling of M41+ and M69+ that were nearly twice those observed in trees grown in elevated CO2 (590 ppmv). However, they also demonstrated the lowest proportion of completely labeled isoprene molecules. These results suggest that under reduced atmospheric CO2 availability, more carbon from stored/older carbon sources is involved in isoprene biosynthesis, and this carbon most likely enters the isoprene biosynthesis pathway through the pyruvate substrate. We offer direct evidence that extra-chloroplastic rather than chloroplastic carbon sources are mobilized to increase the availability of pyruvate required to up-regulate the isoprene biosynthesis pathway when trees are grown in sub-ambient CO2.