Mesophyll conductance (g m) describes the ease with which CO 2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing g m is suggested as a means to engineer increases in photosynthesis by increasing [CO 2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO 2 diffusion across the cell wall and thereby increase g m. Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and g m measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO 2 uptake. Up-regulating CGR3 provides a new strategy for increasing g m in dicotyledonous crops, leading to higher CO 2 assimilation and a potential means to sustainable crop yield improvement.