Abstract: Seedlings of the popular maize cultivar “Zhengdan-958” growing in pots individually were exposed to suitable soil water conditions as control, light water shortage, moderate water shortage, and severe water shortage, corresponding to soil water contents between 75%-85%, 65%-75%, 55%-65%, and 45%-55% of field water capacity, respectively. Responses of stomatal aperture, Kranz anatomy, and vascular bundle structure to different water contents of maize leaves were investigated. Results showed that under increased water shortages, the levels of H₂O₂ in both guard cells and subsidiary cells were enhanced, also the fluorescence intensity of H₂O₂ labeled with fluorescent dye increased, while stomatal aperture and conductance decreased gradually. Moreover, Kranz cells were messily arranged and the cell size became smaller and smaller, and the structure of bundle sheath cells went irregular; and the sectional area of the big bundle and xylem, the cell number of phloem, and the thickness of whole leaf and of upper and lower epidermis reduced. In addition, the number of chloroplasts in mesophyll cells and vascular bundle sheath cells decreased, particularly under the moderate water deficit, chloroplasts in Kranz cells which located in the inside of cytoplasmic membrane and cling on the cell wall spread to the direction of cell center. It demonstrated that stomatal closing of maize could be regulated by H₂O₂ in guard cells and subsidiary cells together, and H₂O₂ in subsidiary cells maybe played a cooperative role. In conclusion, under increased soil water shortages, droughtinduced H₂O₂ accumulations in both guard cells and subsidiary cells of maize leaves participated in the regulation of stomatal closing. And, the size of Kranz cells and bundle sheath cells, the cell number of phloem, and the area of the xylem and phloem reduced, thereby, reducing water shortageinduced damage.