Mesophyll conductance (gm) describes the ease with which CO2passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasinggmhas been suggested as a means to engineer increases in photosynthesis by increasing [CO2] 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 CO2diffusion across the cell wall and thereby increasegm. Across three independent events in tobacco strongly expressingAtCGR3,mesophyll cell wall thickness was decreased by 7-13%, wall porosity increased by 75%, andgmmeasured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2uptake. UpregulatingCGR3provides a new strategy for increasinggmin dicotyledonous crops, leading to higher CO2assimilation and a potential means to sustainable crop yield improvement.