Carbon isotope discrimination (Delta) has been used widely to infer intrinsic water-use efficiency (iWUE) of C-3 plants, a key parameter linking carbon and water fluxes. Despite the essential role of mesophyll conductance (g(m)) in photosynthesis and Delta, its effect on Delta-based predictions of iWUE has generally been neglected. Here, we derive a mathematical expression of iWUE as a function of Delta that includes g(m) (iWUE(mes)) and exploits the g(m)-stomatal conductance (g(sc)) relationship across drought-stress levels and plant functional groups (deciduous or semideciduous woody, evergreen woody and herbaceous species) in a global database. iWUE(mes) was further validated with an independent dataset of online-Delta and CO2 and H2O gas exchange measurements with seven species. Drought stress reduced g(sc) and g(m) by nearly one-half across all plant functional groups, but had no significant effect on the g(sc) : g(m) ratio, with a well supported value of 0.79 +/- 0.07 (95% CI, n = 198). g(m) was negatively correlated to iWUE. Incorporating the g(sc) : g(m) ratio greatly improved estimates of iWUE, compared with calculations that assumed infinite g(m). The inclusion of the g(sc) : g(m) ratio, fixed at 0.79 when g(m) was unknown, proved desirable to eliminate significant errors in estimating iWUE from Delta across various C-3 vegetation types.