Researchers have been examining the biological function(s) of isoprene in isoprene-emitting species for two decades. There is overwhelming evidence that leaf-internal isoprene increases the thermo-tolerance of plants and protects them against oxidative stress, thus mitigating a wide range of abiotic stresses. However, the mechanisms of abiotic stress mitigation by isoprene are still under debate. Here we assessed the impact of isoprene on the emission of NO and S-nitroso-proteome of isoprene-emitting (IE) and non-isoprene-emitting (NE) gray poplar (Populus × canescens (Aiton.) Sm.) after acute ozone fumigation. The short-term oxidative stress induced a rapid and strong emission of NO in NE compared to IE genotypes. Whereas IE and NE plants exhibited under non-stressful conditions only slight differences in their S-nitrosylation pattern, the in vivo S-nitroso-proteome of the NE genotype was more susceptible to ozone-induced changes compared to the IE plants. The results suggest that the nitrosative pressure (NO burst) is higher in NE plants, underlining the proposed molecular dialogue between isoprene and the free radical NO. Proteins belonging to the photosynthetic light and dark reactions, the TCA cycle, protein metabolism, and redox regulation exhibited an increased S-nitrosylation in NE samples compared to IE plants upon oxidative stress. Because the post-translational modification of proteins via S-nitrosylation often impacts enzymatic activities, the present data suggest that isoprene indirectly regulates the production of ROS via the control of the S-nitrosylation level of ROS-metabolizing enzymes, thus modulating the extent and velocity at which the ROS and NO signaling molecules are generated within a plant cell.