Isoprene (C5H8) is a hydrocarbon gas emitted by many tree species and has been shown to protect photosynthesis under abiotic stress. Under optimal conditions for photosynthesis, ~70%–90% of carbon used for isoprene biosynthesis is produced from recently assimilated atmospheric CO2. While the contribution of alternative carbon sources that increase with leaf temperature and other stresses have been demonstrated, uncertainties remain regarding the biochemical source(s) of isoprene carbon. In this study, we investigated leaf isoprene emissions (Is) from neotropical species Inga edulis Mart. as a function of light and temperature under ambient (450 µmol m−2 s−1) and CO2-free (0 µmol m−2 s−1) atmosphere. Is under CO2-free atmosphere showed light-dependent emission patterns similar to those observed under ambient CO2, but with lower light saturation point. Leaves treated with the photosynthesis inhibitor DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) failed to produce detectable Is in normal light under a CO2-free atmosphere. While strong temperature-dependent Is were observed under CO2-free atmosphere in the light, dark conditions failed to produce detectable Is even at the highest temperatures studied (40 °C). Treatment of leaves with 13C-labeled sodium bicarbonate under CO2-free atmosphere resulted in Is with over 50% containing at least one 13C atom. Is under CO2-free atmosphere and standard conditions of light and leaf temperature represented 19% ± 7% of emissions under ambient CO2. The results show that the reassimilation of leaf internal CO2 contributes to Is in the neotropical species I. edulis. Through the consumption of excess photosynthetic energy, our results support a role of isoprene biosynthesis, together with photorespiration, as a key tolerance mechanism against high temperature and high light in the tropics.