Progesterone is an important sex hormone for pregnancy and also has neuroprotective and anticonvulsant effects. It is well-known that full-term parturients become more susceptible to volatile anesthetics. Glutamate transporters are important for preventing neurotoxicity and anesthetic action in the central nervous system. We investigated the effects of progesterone on the activity of glutamate transporter type 3 (EAAT3), the major neuronal EAAT. EAAT3 was expressed in Xenopus laevis oocytes by injecting its mRNA. Oocytes were incubated with diluted progesterone for 72 h. Two-electrode voltage clamping was used to measure membrane currents before, during, and after applying 30 μM L-glutamate. Progesterone (1–100 nM) significantly increased EAAT3 activity in a dose-dependent manner. Our kinetic study showed that the Vmax was increased in the progesterone group compared with that in the control group (2.7±0.2 vs. 3.6±0.2 μC for control group vs. progesterone group; n=18–23; P<0.05), however, Km was unaltered (46.7±10.2 μM vs. 55.9±10.5 μM for control group vs. progesterone group; n=18–23; P>0.05). Phorbol-12-myristate-13-acetate, a protein kinase C (PKC) activator, did not change progesterone-enhanced EAAT3 activity. Inhibitors of PKC or phosphatidylinositol 3-kinase (PI3K) abolished the progesterone-induced increases in EAAT3 activity. Our results suggest that progesterone enhances EAAT3 activity and that PKC and PI3K are involved in mediating these effects. These effects of progesterone may contribute to its anticonvulsant and anesthesia-related properties.