Flower opening is important for successful pollination in many plant species, and some species repeat reversible flower opening and closing movements. This is thought to be due to the turgor pressure change caused by the water influx/efflux, which depends on osmotic oscillation in the cells. In some ornamental plants, it has been suggested that water channel aquaporin may play an important role in flower opening. However, the molecular mechanism(s) involved in flower movement are largely unknown. Using Gentiana flowers, which show reversible movement in response to temperature and light stimuli, as a model, we showed that reversible flower opening is regulated by aquaporin GsPIP2;2 and GsPIP2;7. In particular, phosphorylation of the C-terminal serine residue of GsPIP2;2 is important for the transport activity and correlates closely with the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that reversible flower opening is regulated by GsCPK16-dependent GsPIP2;2 phosphorylation and activation, with stimulus-induced calcium signals acting as triggers. CPK-dependent phosphorylation and activation of PIP2s may be one of the universal regulatory mechanisms for flower opening in plants.