AbstractThe response of mesophyll conductance (gm) to CO2 plays a key role in photosynthesis and ecosystem carbon cycles under climate change. Despite numerous studies, there is still debate about how gm responds to short‐term CO2 variations. Here we used multiple methods and looked at the relationship between stomatal conductance to CO2 (gsc) and gm to address this aspect. We measured chlorophyll fluorescence parameters and online carbon isotope discrimination (Δ) at different CO2 mole fractions in sunflower (Helianthus annuus L.), cowpea (Vigna unguiculata L.), and wheat (Triticum aestivum L.) leaves. The variable J and Δ based methods showed that gm decreased with an increase in CO2 mole fraction, and so did stomatal conductance. There were linear relationships between gm and gsc across CO2 mole fractions. gm obtained from A‐Ci curve fitting method was higher than that from the variable J method and was not representative of gm under the growth CO2 concentration. gm could be estimated by empirical models analogous to the Ball‐Berry model and the USO model for stomatal conductance. Our results suggest that gm and gsc respond in a coordinated manner to short‐term variations in CO2, providing new insight into the role of gm in photosynthesis modelling.