Isoprene is the most abundant volatile compound emitted by vegetation. It influences air chemistry and is thought to take part in plant defense reactions against abiotic stress such as high temperature or ozone. However, whether or not isoprene emission impacts ozone tolerance of plants is still in discussion. In this study, we exploited the transgenic non-isoprene emitting grey poplar (Populus x canescens (Aiton) Sm.) in a biochemical and physiological model study to investigate the effect of acute ozone stress on the elicitation of defense-related emissions of plant volatiles, on photosynthesis and on the antioxidative system. We recorded that non-isoprene emitting poplars were more resistant to ozone as indicated by less damaged leaf area and higher assimilation rates compared to ozone-exposed wild-type (WT) plants. The integral of green leaf volatile emissions was different between the two poplar phenotypes and was a reliable early marker for subsequent leaf damage. For other stress-induced volatiles, such as mono-, homo- and sesquiterpenes and methyl salicylate, similar time profiles, pattern and emission intensities were observed in both transgenic and WT plants. However, unstressed non-isoprene emitting poplars are characterized by elevated levels of ascorbate and alpha-tocopherol as well as by a more effective de-epoxidation ratio of xanthophylls than the WT. Since ozone quenching properties of ascorbate are much higher than those of isoprene and furthermore alpha-tocopherol is also an essential antioxidant, non-isoprene emitting poplars might benefit from changes within the antioxidative system by providing them with enhanced ozone tolerance.