Since gonadal female hormones act on and protect neurons, it is well known that the female brain is less vulnerable to stroke or other brain insults than the male brain. Although glial functions have been shown to affect the vulnerability of the brain, little is known if such a sex difference exists in glia, much less the mechanism that might cause gender-dependent differences in glial functions. In this study, we show that in vitro astrocytes obtained from either female or male pups show a gonadal hormone-independent phenotype that could explain the gender-dependent vulnerability of the brain. Female spinal astrocytes cleared more glutamate by GLAST than male ones. In addition, motoneurons seeded on female spinal astrocytes were less vulnerable to glutamate than those seeded on male ones. It is suggested that female astrocytes uptake more glutamate and reveal a stronger neuroprotective effect against glutamate than male ones. It should be noted that such an effect was independent of gonadal female hormones, suggesting that astrocytes have cell-autonomous regulatory mechanisms by which they transform themselves into appropriate phenotypes.