AbstractThe oxygen isotope composition of cellulose (δ18OCel) archives hydrological and physiological information. Here, we assess previously unexplored direct and interactive effects of the δ18O of CO2 (δ18OCO2), nitrogen (N) fertilizer supply and vapour pressure deficit (VPD) on δ18OCel, 18O‐enrichment of leaf water (Δ18OLW) and cellulose (Δ18OCel) relative to source water, and pexpx, the proportion of oxygen in cellulose that exchanged with unenriched water at the site of cellulose synthesis, in a C4 grass (Cleistogenes squarrosa). δ18OCO2 and N supply, and their interactions with VPD, had no effect on δ18OCel, Δ18OLW, Δ18OCel and pexpx. Δ18OCel and Δ18OLW increased with VPD, while pexpx decreased. That VPD‐effect on pexpx was supported by sensitivity tests to variation of Δ18OLW and the equilibrium fractionation factor between carbonyl oxygen and water. N supply altered growth and morphological features, but not 18O relations; conversely, VPD had no effect on growth or morphology, but controlled 18O relations. The work implies that reconstructions of VPD from Δ18OCel would overestimate amplitudes of VPD variation, at least in this species, if the VPD‐effect on pexpx is ignored. Progress in understanding the relationship between Δ18OLW and Δ18OCel will require separate investigations of pex and px and of their responses to environmental conditions.