Improving photosynthetic efficiency has recently emerged as a promising way to increase crop production in a sustainable manner. While chloroplast size may affect photosynthetic efficiency in several ways, we aimed to explore whether chloroplast size manipulation can be a viable approach to improving photosynthetic performance. Several tobacco (Nicotiana tabacum) lines with contrasting chloroplast sizes were generated via manipulation of chloroplast division genes to assess photosynthetic performance under steady-state and fluctuating light. A selection of lines was included in a field trial to explore productivity. Lines with enlarged chloroplasts underperformed in most of the measured traits. Lines with smaller and more numerous chloroplasts showed a similar efficiency compared with wild-type (WT) tobacco. Chloroplast size only weakly affected light absorptance and light profiles within the leaf. Increasing chloroplast size decreased mesophyll conductance (g ) but decreased chloroplast size did not increase g . Increasing chloroplast size reduced chloroplast movements and enhanced non-photochemical quenching. The chloroplast smaller than WT appeared to be no better than WT for photosynthetic efficiency and productivity under field conditions. The results indicate that chloroplast size manipulations are therefore unlikely to lead to higher photosynthetic efficiency or growth. [Abstract copyright: © 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.]