Stomata play critical roles in drought tolerance formation by regulating the stomata movement, which is essential for balancing plant photosynthesis and transpiration. Drought priming have been convinced to improve drought tolerance by improving photosynthesis and water use efficiency. However, little is known about how the dynamic stomatal behavior during priming-recovery-drought stress-recovery cycle, which may relate to dynamic behavior of stomata to make the tradeoff between photosynthesis and transpiration in wheat. With portable non-invasive guard cell dynamic measurement, the results found that drought priming increased stomatal sensitivity, specifically, that drought priming promotes stomatal closure faster under drought stress, and stomatal reopening faster during recovery. In-stiu ion flux and related channel expression of guard cells results found that compared with non-primed plants, drought priming induced the higher accumulation of guard cell ABA, which activating the Ca2+ influx channel with induced the guard cell Ca2+ influx, and also activating the Ca2+ signaling pathway. The guard cell K+ outward channel was activated, with genes encoding anion channel were higher expressed, which all lead to the enhanced K+ efflux, and then quicker promote stomatal closure in primed plants under drought stress. Our findings suggest a novel observation and underlying mechanisms of dynamic stomatal behavior in intact wheat leaves under drought priming-recovery-drought stress-recovery cycle. The results will deeply illustrate the underlying mechanisms of stomatal regulation in improved drought tolerance of wheat, and help breeding new drought resistant wheat cultivar and providing theoretical and technical support for the construction of drought-resistant cultivation techniques.