Transpiration (T) is perhaps the largest fluxes of water from the land surface to the atmosphere and is susceptible to changes in climate, land use and vegetation structure. However, predictions of future transpiration fluxes vary widely and are poorly constrained. Stable water isotopes can help expand our understanding of land–atmosphere water fluxes but are limited by a lack of observations and a poor understanding of how the isotopic composition of transpired vapour (δT) varies. Here, we present isotopic data of water vapour, terrestrial water and plant water from a deciduous forest to understand how vegetation affects water budgets and land–atmosphere water fluxes. We measured subdiurnal variations of δ18OT from three tree species and used water isotopes to partition T from evapotranspiration (ET) to quantify the role of vegetation in the local water cycle. We find that δ18OT deviated from isotopic steady‐state during the day but find no species‐specific patterns. The ratio of T to ET varied from 53% to 61% and was generally invariant during the day, indicating that diurnal evaporation and transpiration fluxes respond to similar atmospheric and micrometeorological conditions at this site. Finally, we compared the isotope‐inferred ratio of T to ET with results from another ET partitioning approach that uses eddy covariance and sap flux data. We find broad midday agreement between these two partitioning techniques, in particular, the absence of a diurnal cycle, which should encourage future ecohydrological isotope studies. Isotope‐inferred estimates of transpiration can inform land surface models and improve our understanding of land–atmosphere water fluxes.