Leaf water gets isotopically enriched through transpiration, and diffusion of enriched water through the leaf depends on transpiration flow and the effective path length (L). The aim of this work was to relate L with physiological variables likely to respond to similar processes. We studied the response to drought and vein severing of leaf lamina hydraulic conductance (Klamina), mesophyll conductance for CO2 (gm) and leaf water isotope enrichment in Vitis vinifera L cv. Grenache. We hypothesized that restrictions in water pathways would reduce Klamina and increase L. As a secondary hypothesis, we proposed that, given the common pathways for water and CO2 involved, a similar response should be found in gm. Our results showed that L was strongly related to mesophyll variables, such as Klamina or gm across experimental drought and vein-cutting treatments, showing stronger relationships than with variables included as input parameters for the models, such as transpiration. Our findings were further supported by a literature survey showing a close link between L and leaf hydraulic conductance (Kleaf = 31.5 × L−0.43, r2 = 0.60, n = 24). The strong correlation found between L, Klamina and gm supports the idea that water and CO2 share an important part of their diffusion pathways through the mesophyll.