The objective of this study is to globally assess the effects of atmospheric nitrogen deposition and climate, associated with rising levels of atmospheric CO2, on the variability of carbon isotope discrimination (Δ13C), and intrinsic water-use efficiency (iWUE) of angiosperm and conifer tree species. Eighty-nine long-term isotope tree-ring chronologies, representing 23 conifer and 13 angiosperm species for 53 sites worldwide, were extracted from the literature, and used to obtain long-term time series of Δ13C and iWUE. Δ13C and iWUE were related to the increasing concentration of atmospheric CO2 over the industrial period (1850–2000) and to the variation of simulated atmospheric nitrogen deposition and climatic variables over the period 1950–2000. We applied generalized additive models and linear mixed-effects models to predict the effects of climatic variables and nitrogen deposition on Δ13C and iWUE. Results showed a declining Δ13C trend in the angiosperm and conifer species over the industrial period and a 16.1% increase of iWUE between 1850 and 2000, with no evidence that the increased rate was reduced at higher ambient CO2 values. The temporal variation in Δ13C supported the hypothesis of an active plant mechanism that maintains a constant ratio between intercellular and ambient CO2 concentrations. We defined linear mixed-effects models that were effective to describe the variation of Δ13C and iWUE as a function of a set of environmental predictors, alternatively including annual rate (Nrate) and long-term cumulative (Ncum) nitrogen deposition. No single climatic or atmospheric variable had a clearly predominant effect, however, Δ13C and iWUE showed complex dependent interactions between different covariates. A significant association of Nrate with iWUE and Δ13C was observed in conifers and in the angiosperms, and Ncum was the only independent term with a significant positive association with iWUE, although a multi-factorial control was evident in conifers.