Mesophyll conductance is the CO2 diffusion efficiency through the leaf from the substomatal cavity to chloroplasts located in plant cells. It is an important step determining photosynthetic efficiency next to photosynthetic biochemistry and stomatal conductance. Mesophyll conductance limits photosynthesis and therefore plant growth. Hence, ignoring mesophyll conductance in vegetation models results in mistakes in estimating the carbon cycle. Gymnosperms were so far the most understudied plant group in terms of photosynthetic limitations and mesophyll conductance, despite the relatively higher importance of these factors in evolutionarily older plant groups like gymnosperms. Furthermore, gymnosperms are ecologically, economically and evolutionarily highly significant. For example, 40% of all forests are estimated to consist of gymnosperms. I studied the role of mesophyll conductance in limiting photosynthesis and its underlying foliage anatomical characteristics in gymnosperms. Gymnosperms had lower average mesophyll conductance than angiosperms and it was the key limiter of photosynthesis across the sample. The mesophyll conductance of gymnosperms was mainly determined by the abundance of chloroplasts lining the cell walls facing the intercellular airspace and mesophyll cell wall thickness, where species that had more chloroplasts lining the intercellular airspace and thinner cell walls also had higher mesophyll conductance. The share of photosynthetic limitation by mesophyll compared to stomata and biochemistry depended on foliage shape. Gymnosperms resembled evolutionarily older groups like horsetails in some photosynthetic traits, while in others they resembled robust-leaved angiosperms adapted to dry environments. However, the combined physiology and anatomy traits illustrate a unique set of adaptations in gymnosperms proving that the role of mesophyll conductance and the effect of its underlying structural traits cannot be simply generalized across different plant clades. It has been suggested that the lower mesophyll conductance and the anatomical traits determining it give gymnosperms and advantage over angiosperms in the changing climate.