Finite mesophyll conductance (gm) reduces the rate of CO2 diffusion from the leaf intercellular space to the chloroplast and constitutes a major limitation of photosynthesis in trees. While it is well established that gm is decreased by stressors such as drought and high temperature, few studies have investigated if the phytotoxic air pollutant ozone (O3) affects gm. We quantified the relative importance of three different types of limitations of photosynthesis in poplar trees exposed to elevated O3: decreases in stomatal conductance, gm and biochemical photosynthetic capacity. The O3-induced reductions in light-saturated net photosynthesis were linked to significant declines in gm and biochemical photosynthetic capacity (in particular carboxylation). There was no significant effect of O3 on stomatal conductance. Of the O3-induced limitations on photosynthesis, gm limitation was by far the most important (−16%) while biochemical limitation (−8%) was rather small. Both limitations grew in magnitude over the study period and varied in response to leaf-specific O3 exposure. Our findings suggest that declines in gm may play a key role in limiting photosynthesis of plants exposed to elevated O3, an effect hitherto overlooked.