Glioblastoma is the most aggressive form of primary brain tumor with a tendency to invade surrounding healthy brain tissues, rendering tumors of this type largely incurable. Aquaporin-4 (AQP4) is a key molecule involved in maintaining water and ion homeostasis in the central nervous system and has been recently reported to play a role in cell migration in addition to its well-known function in brain edema. Increased AQP4 expression has been demonstrated in glioblastoma multiforme (GBM), suggesting that it is also involved in malignant brain tumors. Here, we identify a novel role for aquaporin-4 in glioblastoma cell migration and invasion. In the present study, we used small-interference RNA technology and a pharmacological inhibitor to knock down the expression of AQP4, which resulted in specific and massive impairment of glioblastoma cell migration and invasion in vitro and in vivo. In addition, we demonstrated the possible mechanisms by which AQP4 functions in the process of glioblastoma cell invasion. The downregulation of matrix metalloprotease-2 (MMP-2) expression in LN229 cells with AQP4 reduction coincided with decreased cell invasive ability. Furthermore, our study showed that AQP4 may also be involved in the regulation of glioblastoma cell adhesion. The expression of β-catenin and connexin 43 were increased in AQP4-downregulated LN229 cells consistent with their enhanced cell-cell adhesion ability. In summary, our results indicate that AQP4 is involved in the control of glioblastoma cell migration and invasion and may be a potential therapeutic target for glioblastoma cell infiltration.