Astrocytes are active elements of the brain circuitry. They integrate neuronal signals, exhibit Ca++ excitability and process information. Ca++ signaling in activated astrocytes has been proposed to trigger the release of many neuroactive molecules, such as glutamate, ATP and D-serine, which can modulate neuronal excitability, synaptic activity and plasticity. Aquaporin-4 (AQP4) is strongly expressed in astrocyte endfeet and has an important role in brain water flux at the blood-brain and CSF-brain barriers. In the present study we have used WT and AQP4 KO astrocyte primary cultures to show that AQP4 plays a role in glial calcium homeostasis. By Ca++ imaging experiments we demonstrated that under hypotonic stress WT astrocytes exhibited an intracellular Ca++ increase whose amplitude was 6 times higher in the presence of external Ca++, indicating a Ca++ influx from the extracellular and not intracellular stores. The same experiments, performed in parallel on AQP4 KO astrocytes, showed that the amplitude of this phoenomenon was significantly reduced and associated to a delay in calcium influx, suggesting a direct effect of the altered water permeability on the hypotonic shock dependent Ca++ increase. Gadolinium and Ruthenium red were later used to show that this effect was dependent on plasma membrane stretch activated Ca++ channels. Finally, the use of CPA helped us demonstrating that the difference in the influx of calcium was not due to ICrac but mainly related to the magnitude of membrane stretch. All together these findings suggest that AQP4 plays a pivotal role in astrocyte Ca++ homeostasis and is therefore involved in the modulation of neuronal excitability, synaptic activity and plasticity.