Ma, Wei TingTcherkez, GuillaumeWang, Xu MingSchaufele, RudiSchnyder, HansYang, YushengGong, Xiao Ying2022-07-280028-646Xhttp://hdl.handle.net/1885/270000Carbon 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 semideciduous 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 and gm by nearly one‐half across all plant functional groups, but had no significant effect on the gsc : gm ratio, with a well supported value of 0.79 ± 0.07 (95% CI, n = 198). gm was negatively correlated to iWUE. Incorporating the gsc : gm ratio greatly improved estimates of iWUE, compared with calculations that assumed infinite gm. The inclusion of the gsc : gm ratio, fixed at 0.79 when gm was unknown, proved desirable to eliminate significant errors in estimating iWUE from Δ across various C3 vegetation types.This work was supported by the National Natural Science Foun-dation of China (NSFC 31870377), the Guangdong NaturalScience Foundation (2018A030313450) and the DeutscheForschungsgemeinschaft (SCHN 557/7-1 and SCHN 557/9-1)application/pdfen-AU© 2020 The authors13C discriminationdrought stressmesophyll conductancephotosynthesisstable isotopestomatal conductancetranspirationwater-use efficiency.202110.1111/nph.169582021-08-01