Background: Plants respond to changes in vapour pressure deficit ( VPD) between the leaf and the atmosphere through changes in stomatal response, which can consequently affect transpiration, photosynthesis, and leaf-level water use efficiencies. With projected warmer air temperatures, changes in rainfall distribution and altered VPD in future climates, it is important to understand the potential effect of VPD on leaf-level physiology of field-grown crops. The aim of this study was to assess the impact of altered VPD on leaf-level physiology of field-grown cotton to improve the current understanding of the plant-by-environment interaction, thereby contributing to validation and improvement of physiological and yield response models. Different VPD environments in the field were gener-ated by planting cotton on three dates within the sowing window (early-season (S1) = 5th October 2011; mid-season (S2) = 9th November 2011; and late-season (S3) = 30th November 2011). VPD was also modified by altering crop irrigations. Results: VPDL accounted for the largest proportion of the explained variation in both stomatal conductance (32％～39％) and photosynthetic (16％～29％) responses of cotton. Generally, smaller percentages of variation were attributed to other main factors such as the individual plant (Plant), and accumulated temperature stress hours (ASH; a measure of plant water status over time) and interactive factors, including leaf vapour pressure deficit -( VPDL) × Plant and Plant × ASH; however, a proportion of variation was unexplained. In addition, the Asat/E (instantaneous transpira-tion efficiency, ITE) model developed based on cotton grown in the glasshouse was applied to cotton grown in the field. We found that the modelled Asat/E and field-measured Asat/E were very similar, suggesting that the mechanistic basis for ITE was similar in both environments. Conclusions: This study highlights the importance of accounting for VPD in climate change research, given that stomata are highly responsive to changes in VPD. This experiment provides a basis for physiology and production models, particularly in terms of cotton response to projected climatic environments.