Increasing stomatal movement is beneficial to improve plant water use efficiency and drought resilience. Contradictory results indicate that aquaporins might regulate stomatal movement. Here, we tested whether the maize plasma membrane PIP2;5 aquaporin affects stomatal closure under water deficit, abscisic acid (ABA) or vapour pressure deficit (VPD) treatment in intact plants, detached leaves or peeled epidermis. Transpiration, stomatal conductance (g(s)) and aperture and reactive oxygen species (ROS) in stomatal complexes were studied in maize lines with increased or knocked down (KD) PIP2;5 gene expression. In well-watered conditions, the PIP2;5 overexpressing (OE) plants transpired more than wild types (WTs), while no significant difference in transpiration was observed between pip2;5 KD and WT. Upon mild water deficit or low ABA concentration treatments, transpiration and g(s) decreased more in PIP2;5 OE lines and less in pip2;5 KD lines, in comparison with WTs. In the detached epidermis, ABA treatment induced faster stomatal closing in PIP2;5 OE lines compared to WTs, while pip2;5 KD stomata were ABA insensitive. These phenotypes were associated with guard cell ROS accumulation. Additionally, PIP2;5 is involved in the transpiration decrease observed under high VPD. These data indicate that maize PIP2;5 is a key actor increasing the sensitivity of stomatal closure to water deficit.