Oxygen isotopic composition (Δ18OLW) of leaf water can help improve our understanding of how anatomy interacts with physiology to influence leaf water transport. Leaf water isotope models of Δ18OLW such as the Péclet effect model have been developed to predict Δ18OLW, and it incorporates transpiration rate (E) and the mixing length between unenriched xylem water and enriched mesophyll water, which can occur in the mesophyll (Lm) or veins (Lv). Here we used two cell wall composition mutants grown under two light intensities and RH to evaluate the effect of cell wall composition on Δ18OLW. In maize (Zea mays), the compromised ultrastructure of the suberin lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL TRANSFERASE mutant (Zmasft) reduced barriers to apoplastic water movement, resulting in higher E and Lv and, consequently, lower Δ18OLW. In cellulose synthase-like F6 (Cslf6) mutants and wildtype of rice (Oryza sativa), the difference in Δ18OLW in plants grown under high and low growth light intensity co-varied with their differences in stomatal density. These results show that cell wall composition and stomatal density influence Δ18OLW by altering the Péclet effect and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model.