Controversial evidence of CO2-responsiveness of isoprene emission has been reported in the literature with the response ranging from inhibition to enhancement, but the reasons for such differences are not understood. We studied isoprene emission characteristics of hybrid aspen (Populus tremula x P. tremuloides) grown under ambient (380 μmol mol−1) and elevated (780 μmol mol−1) [CO2] to test the hypothesis that growth [CO2] effects on isoprene emission are driven by modifications in substrate pool size, reflecting altered light use efficiency for isoprene synthesis. A novel in vivo method for estimation of the pool size of the immediate isoprene precursor, dimethylallyldiphosphate (DMADP) and the activity of isoprene synthase was used. Growth at elevated [CO2] resulted in greater leaf thickness, more advanced development of mesophyll and moderately increased photosynthetic capacity due to morphological “upregulation”, but isoprene emission rate under growth light and temperature was not significantly different among ambient- and elevated-[CO2]-grown plants independent of whether measured at 380 μmol mol−1 or 780 μmol mol−1 CO2. However, DMADP pool size was significantly less in elevated-[CO2]-grown plants, but this was compensated by increased isoprene synthase activity. Analysis of CO2 and light response curves of isoprene emission demonstrated that the [CO2] for maximum isoprene emission was shifted to lower [CO2] in elevated-[CO2]-grown plants. The light-saturated isoprene emission rate (Imax,Q) was greater, but the quantum efficiency at given Imax,Q was less in elevated-[CO2]-grown plants, especially at higher CO2 measurement concentration, reflecting stronger DMADP limitation at lower light and higher [CO2]. These results collectively demonstrate important shifts in light and CO2-responsiveness of isoprene emission in elevated-[CO2]-acclimated plants that need consideration in modeling isoprene emissions in future climates.