Stomata regulate CO 2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (g sw ). However, the quantitative relationship between reduced SD, g sw , and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEPF2) in sugarcane reduced SD by 26-38% but did not affect g sw compared with the wild type. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO 2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum g sw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining g sw when SD is reduced. Meta-analysis across C 3 dicots, C 3 monocots, and C 4 monocots revealed that engineered reductions in SD are strongly correlated with lower g sw (r 2 =0.60-0.98), but this response is damped relative to the change in anatomy.