Abstract Decline in mesophyll conductance ( g m ) plays a key role in limiting photosynthesis in plants exposed to elevated ozone (O 3 ). Leaf anatomical traits are known to influence g m , but the potential effects of O 3 ‐induced changes in leaf anatomy on g m have not yet been clarified. Here, two poplar clones were exposed to elevated O 3 . The effects of O 3 on the photosynthetic capacity and anatomical characteristics were assessed to investigate the leaf anatomical properties that potentially affect g m . We also conducted global meta‐analysis to explore the general response patterns of g m and leaf anatomy to O 3 exposure. We found that the O 3 ‐induced reduction in g m was critical in limiting leaf photosynthesis. Changes in liquid‐phase conductance rather than gas‐phase conductance drive the decline in g m under elevated O 3, and this effect was associated with thicker cell walls and smaller chloroplast sizes. The effects of O 3 on palisade and spongy mesophyll cell traits and their contributions to g m were highly genotype‐dependent. Our results suggest that, while anatomical adjustments under elevated O 3 may contribute to defense against O 3 stress, they also cause declines in g m and photosynthesis. These results provide the first evidence of anatomical constraints on g m under elevated O 3 .