SummaryContradictory results indicate that aquaporins might facilitate the diffusion of both water and H2O2 during abscisic acid (ABA) triggered stomatal closure. Here, we tested whether maize plasma membrane PIP2;5 aquaporin regulates stomatal closure under water deficit or ABA treatment in intact plants, detached leaves, and peeled epidermis.Transpiration, stomatal conductance and aperture, as well as reactive oxygen species (ROS) in stomatal complexes were studied in maize lines deregulated in PIP2;5 gene expression, under water deficit and/or ABA treatments.In well-watered conditions, the PIP2;5 overexpressing (OE) plants transpired more than the wild-type plants (WT), while no significant difference in transpiration was observed between pip2;5 KO and WT plants. Upon mild-water deficit or low ABA concentration treatment, the transpiration and stomatal conductance decreased more in PIP2;5 OE, and less in pip2;5 KO lines, in comparison with WT plants. Using isolated epidermis, ABA treatment induced faster stomatal closing in PIP2;5 OE lines compared to the WT, while pip2;5 KO stomata were ABA insensitive. These phenotypes were associated with guard cell ROS accumulation.Together, these data indicate that maize PIP2;5 regulates early stomatal closure for water conservation upon a water deficit environment.