Data were used to investigate long-term changes in tree intrinsic water use efficiency (iWUE, i.e., the ratio between CO2 assimilation and stomatal conductance) and the underlying physiological mechanisms. We used d18O to estimate the 18O enrichment in leaf water above the source water, d18OLW. Moreover we assessed the relationship between isotope-derived parameters and atmospheric CO2 (ca) and climate factors. Isotope-related parameters included in the dataset are: a-cellulose d13C, carbon isotope discrimination (d13C), leaf internal CO2 concentration (ci) and the leaf to atmospheric CO2 concentrations (ci/ca), a-cellulose d18O, estimated d18O in precipitation (see Method), oxygen isotope discrimination above the source water (d18O). The dataset includes also the following climate parameters: growing season temperature (Tgrs), precipitation (Pgrs) and vapor pressure deficit (VPDgrs) and mean annual temperature (Ta), precipitation (Ta) and vapor pressure deficit (VPDa), and standard precipitation-evaporation index, SPEI, relative to August, with 3 months lag from the global database. Finally, we also include the ca values that were used to calculate d13C, iWUE and ci/ca. All the equations used to calculate the isotope-derived parameters, including the leaf water d18O (see Figure 3 in Guerrieri et al. 2019 PNAS) are also provided.