Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels (KATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus KATP channel openers (KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that KATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters (GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium (MPP+). These potentiated effects were completely abolished by mitochondrial KATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C (PKC). These findings are the first to demonstrate that activation of KATP channel, especially mitochondrial KATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.