Aquaporin-4 (AQP4), a water channel protein, is abundantly expressed in astrocytes and plays a key role in the development of brain edema. However, it is not clear whether AQP4 contributes to astrocytic swelling in hypoxia–ischemia (HI). To investigate the roles of AQP4 in astrocytic swelling during HI and reoxygenation, we measured AQP4 expression and astrocytic cellular volume in cultured rat astrocytes following HI and reoxygenation. RNA interference was used to knockdown AQP4 expression (AQP4−/−). Real-time polymerase chain reaction and Western blot analysis were used to detect the inhibitory efficiency of AQP4. We found that the maximal inhibition of AQP4 mRNA and protein in astrocytes after AQP4 siRNA transfection (AQP4−/−) was ∼77 and 85%, respectively, compared to wild-type AQP4 (AQP4+/+) expression. Cellular volume in both AQP4−/− and AQP4+/+ astrocytes was significantly increased during HI compared to cells cultured in normoxia (P < 0.05). However, cellular volume during HI in AQP4−/− astrocytes was significantly less than that in AQP4+/+ astrocytes (P < 0.05). After reoxygenation, the cellular volume gradually decreased to control levels at 7 days in AQP4−/− but at 5 days in AQP4+/+ astrocytes. The different roles of AQP4 during HI and reoxygenation suggest that AQP4 knockdown may protect against water influx in the formation of astrocyte swelling during HI, and may also delay water clearance in the resolution of astrocyte swelling during reoxygenation. In conclusion, AQP4 mediates bidirectional transport of water across astrocytes during HI and reoxygenation. AQP4 manipulation may serve as a novel therapeutic strategy during different periods of hypoxic–ischemic brain edema in neonates. © 2007 Wiley-Liss, Inc.