Land plants regulate their photosynthesis and water transpiration by exchanging gases (CO₂ and H₂O_vapour) with the atmosphere. These exchanges take place through microscopic valves, called stomata, on the leaf surface. The opening of the stomata is regulated by two guard cells that actively and reversibly modify their turgor pressure to modulate the opening of the stomatal pores. Stomatal function depends on the regulation of the ion transport capacities of cell membranes as well as on the modification of the subcellular organisation of guard cells. Here we report how the vacuolar and cytosolic compartments of guard cells quantitatively participate in stomatal opening. We used a genetically encoded biosensor to visualise changes in ionic concentration during stomatal opening. The 3D reconstruction of living guard cells shows that the vacuole is the responsible for the change in guard cell volume required for stomatal opening.