Astrocytes serve various important functions in the CNS, but the molecular mechanisms of their generation and maturation are still enigmatic. Here, we show that Pax6, a key transcription factor that controls neurogenesis, also regulates proliferation, differentiation, and migration of astrocytes in the CNS. We first reveal that Pax6 is expressed in astrocytes during development as well as postnatally in the wild-type mouse. Astrocytes derived from Pax6 homozygous mutants (Sey/Sey) mice exhibited aberrant proliferation together with immature differentiation, both in vivo and in vitro, with higher migration potential in scratch-wound assays in vitro. Furthermore, a larger population of Sey/Sey astrocytes expresses neural stem cell markers such as nestin, Sox2, and prominin-1. These phenotypes of Pax6-deficient astrocytes putatively occur via higher Akt activity. Thus, the breakdown of Pax6 function induces the retention of neural stem-like characteristics and inhibits astrocyte maturation.