The aim of this study was to investigate how alternative irrigation strategies affected grapevine (Vitis vinifera L.) stomatal response to atmospheric vapour pressure deficit (VPD). In two sites, application of partial rootzone drying (PRD) at 90–100% of crop evapotranspiration (ETc) increased stomatal sensitivity of Shiraz (Syrah) grapevines to high VPD compared with control vines irrigated with the same amount of water but applied on both sides of the vine. PRD significantly reduced vine water use (ESF) measured as sap flow and in dry conditions increased the depth of water uptake from the soil profile. In both experiments, PRD reduced vine water use by up to 50% at moderate VPD (~3 kPa) compared with control vines irrigated at the same level. In the same vines, the response to PRD applied at 100% ETc and deficit irrigation applied at 65% ETc was the same, increasing stomatal sensitivity to VPD and decreasing sap flow. Hydraulic signalling apparently did not play a role in changing stomatal sensitivity as there was no difference in stem water potentials between any of the treatment (PRD and DI) and control vines. This suggests that a long distance root-based chemical signal such as ABA may be responsible for the changes in stomatal behaviour. Shiraz grapevines have previously been classified as anisohydric-like, but application of PRD and DI increased stomatal closure in response to conditions of high evaporative demand making the vines behave in a more isohydric-like manner.