Acute administration of opioids produces analgesia, while chronic administration induces tolerance and dependence. Aquaporin 4 (AQP4) is most strongly expressed in astrocytes throughout central nervous system, and plays an important role in some pathophysiological processes in brain. However, whether AQP4 modulates opioid analgesia, tolerance and dependence or not remains unknown. In the present study, the effects of AQP4 deficiency on morphine analgesia, tolerance and physical dependence were investigated. (1) In hot-plate tests, ED(50) values of morphine analgesia were 3.77 and 3.96 mg/kg in male and female AQP4 knockout mice, which were lower than that in wild-type mice (5.23 and 5.20mg/kg in males and females). (2) Repeated treatment with morphine resulted in analgesic tolerance to morphine in wild-type mice, whereas the morphine tolerance was attenuated in AQP4 knockout mice treated as the same schedule. (3) After repeated morphine administration, naloxone precipitation induced significant abstinent jumping in wild-type mice, whereas naloxone-induced abstinent jumping was not observed in AQP4 knockout mice. This suggested that AQP4 deficiency inhibited the development of morphine physical dependence. (4) Repeated morphine administration down-regulated cerebral glutamate transporter 1 (GLT-1) expression in wild-type mice. However, the down-regulation of GLT-1 expression diminished in AQP4 knockout mice. Taken together, these results demonstrated that AQP4 deficiency potentiated morphine analgesia, attenuated morphine tolerance and physical dependence. The suppression of down-regulation of cerebral GLT1 expression might mediate the attenuation of AQP4 deficiency to morphine tolerance and dependence.