There are increasing evidences showing that mesophyll conductance for CO₂ diffusion (g_m) limits leaf photosynthesis as well as, or even more than, stomatal conductance (g_s). Although g_m was widely reported for seed plants, g_m of ferns was reported limitedly. We selected five temperate fern species to estimate their g_m using stable carbon isotope method. Then, the mesophyll anatomies of fronds were observed using light microscope and transmission electron microscope to estimate their effects on g_m. If our data were plotted together with the data of seed plant in the previous studies, g_m values were positively correlated with Sc, surface area of chloroplast facing to interceller air space, while they were negatively correlated with cell wall thickness, which suggests that low Sc with large cell wall thickness of ferns was important factors that explain why g_m of ferns is lower than those of seed plants. Thereafter, the response of g_m and g_s to changing atmospheric CO₂ was observed. Ferns lacked g_s response to CO₂. The g_m of ferns responded more rapidly to changes in CO₂ than g_s, which is similar to seed plants. These results suggest that mechanism for g_m response to CO₂ may be different from that for g_s response to CO₂.