Stomata is the main channel for CO2 to enter into leaf mesophyll cells，and its aperture plays a key role in photosynthetic rate. Previous studies have shown that 5-aminolevulinic acid（ALA）is involved in stomatal regulation in apple leaves，but the underlying mechanism has not been fully elucidated. In the present work，the effects of ALA on cytoplasmic pH and stomatal movement were studied with the abaxial epidermis of leaves of Fuji apple（Malus × domenstica Borkh）. Results showed that when stomatal closure was induced by ABA and benzylamine，the fluorescence intensity of cytoplasmic pH and reactive oxygen species（ROS）increased significantly. When ALA and butyrate were used to inhibit ABA-induced stomatal closure，they also inhibited ABA-induced increase of cytoplasmic pH and ROS fluorescence intensity. Benzylamine weakened the effect of ALA on the stomatal closure induced by ABA，while ALA and butyrate inhibited stomatal closure induced by exogenous H2O2 and Ca2+. These results suggest that cytoplasmic pH is involved in stomatal regulation at the upstream of the signal pathway of ALA regulating stomatal movement. Analysis of qRT-PCR showed that ABA induced up-expressions of Mdvha-c2 and Mdvha-c3 which encode subunits of tonoplast H+-ATPase，whereas ALA inhibited this effect. These suggest that cytosolic acidification induced by ALA may be related to the decrease of ion pump activity in the vacuolar membrane. Based on the above results，the authors conclude that the effect of ALA on stomatal movement in apple leaves is mediated by cytoplasmic pH pathway.