In C3 leaves, diffusion of CO2 into leaves is restricted by stomata and subsequently by the intercellular airspaces and liquid phase into chloroplasts. This diffusion restriction within leaves has been termed mesophyll conductance, gm, which has garnered attention because gm limits photosynthesis at ambient CO2. Recently, some reports have shown that gm varies with CO2 concentration and it may be caused by gating of aquaporins (cooporins) which are located in the plasma membrane and inner envelope of chloroplasts. However, there is still controversy about whether gm responds to CO2 concentration. In this study, the effects of CO2 concentration on gm were examined in tobacco, Arabidopsis and wheat leaves by combining gas exchange with carbon isotope discrimination measurements using tunable diode laser absorption spectroscopy. CO2 was initially increased from 200 to 1,000 ppm and then decreased stepwise to 200 ppm and increased stepwise back to 1000 ppm. In 2% O2, a step increase from 200 to 1,000 ppm significantly decreased gm by 26%–40% in all three species. The CO2 response of gm was less in 21% O2.