Glial water channel aquaporin-4 (AQP4) plays an important role in neuroexcitation phenotypes that are highly associated with potassium homeostasis in brain extracellular space (ECS). The mechanism of how AQP4 modulate the neuroexcitation through potassium redistribution during the neuronal signal transduction remains unknown. Cortical spreading depression (CSD) is a self-propagating wave of neuronal depolarization with increased extracellular potassium concentration ([K+]o), astrocyte swelling and subsequent severe contraction of ECS which provide a model for the mechanism study. Here we characterized the CSD induced by KCl application in wild type (WT) and AQP4 deficient mice (AQP4-/-) and found AQP4-/- mice had a significant decrease in the frequency (6.9 ± 0.3 vs. 10.2 ± 0.5 CSDs/hr; p < 0.01), as well as the propagation velocity of CSD (2.9 ± 0.1 vs. 3.7 ± 0.1 mm/min; p < 0.05). During CSD, the dynamic changes of extracellular potassium were determined using K+-selective microelectrodes and the extracellular space (ECS) was measured by TMA+ method. We found the rate of K+ release and clearance was significantly prolonged in the AQP4-/- mice (t1/2, 10.2 ± 1.8 sec and 43.2 ± 2.3sec) compared to their WT counterparts (t1/2, 7.4 ± 0.3 sec and 35.7 ± 1.0 sec), which were paralleled by a significantly delayed contraction and recovery of ECS to baseline in AQP4-/- mice. There is no difference in baseline or peak [K+]o. Importantly, no alterations were found in the expression or localization of inwardly rectifying K+ channel Kir4.1, gap junction hemichannel connexin-43, and anchor protein alpha-syntrophin in AQP4-/- mice. A computer geometrical modeling of potassium accumulation and clearance during CSD confirmed the major role of ECS dynamic change in modulation of potassium kinetics. These results implicated an essential role of AQP4 in dynamic changes of ECS during CSD, which may be a new mechanism underlying the potassium kinetics and neuroexcitation.