Summary Past studies have established mesophyll diffusion conductance to CO 2 ( g m ) as a variable and significant limitation to plant photosynthesis under steady‐state conditions. However, the role of g m in influencing photosynthesis ( A ) during the transient period of light induction is largely unknown. We combined gas exchange measurements with laser‐enabled carbon isotope discrimination measurements to assess g m during photosynthetic induction, using Arabidopsis as the measurement species. Our measurements revealed three key findings: (1) we found that the rate at which g m approached steady state during induction was not necessarily faster than the induction rate of the carboxylation process, contradictory to what has been suggested in previous studies; (2) g m displayed a strong and consistent coordination with A under both induction and steady‐state settings, hinting that the mechanism driving g m – A coupling does not require physiological stability as a prerequisite; and (3) photosynthetic limitation analysis of our data revealed that when integrated over the entire induction period, the relative limitation of A imposed by g m can be as high as > 35%. The present study provides the first demonstration of the important role of g m in limiting CO 2 assimilation during photosynthetic induction, thereby pointing to a need for more research attention to be devoted to g m in future induction studies.