SummaryThe carbon isotope discrimination (Δ) has been used widely to infer intrinsic water-use efficiency (iWUE) of C3 plants, a key parameter linking carbon and water fluxes. Despite the essential role of mesophyll conductance (gm) in photosynthesis and Δ, its effect on Δ-based predictions of iWUE has generally been neglected.Here, we derive a mathematical expression of iWUE as a function of Δ that includes gm (iWUEmes) and exploits the gm-stomatal conductance (gsc) relationship across drought-stress levels and plant functional groups (deciduous or semi-deciduous woody, evergreen woody and herbaceous species) in a global database. iWUEmes was further validated with an independent dataset of online-Δ and CO2 and H2O gas exchange measurements with seven species.Drought stress reduced gsc by 52% and gm by 45% averaged over all plant functional groups, but had no significant effect on the gsc/gm ratio, suggesting a well-constrained gsc/gm ratio of 0.79±0.07 (95%CI, n=198) across plant functional groups and drought-stress treatments. Due in part to the synchronous behavior of gsc and gm, gm was negatively correlated to iWUE. Incorporating the gsc/gm ratio in the iWUEmes model significantly improved the estimation of iWUE compared to the simple model.The inclusion of gm effects, even using a fixed gsc/gm ratio of 0.79 when gm is unknown, proved desirable to eliminate significant bias in estimating iWUE from Δ across various C3 vegetation types.