Glial fibrillary acidic protein (GFAP) is an enigmatic protein; it currently has no unambiguously defined role. It is expressed in the cytoskeleton of astrocytes in the mammalian brain. We have used co-immunoprecipitation to identify in vivo binding partners for GFAP in the rat and pig brain. We demonstrate interactions between GFAP, the glutamate transporter GLAST, the PDZ-binding protein NHERF1, and ezrin. These interactions are physiologically relevant; we demonstrate in vitro that transport of D-aspartate (a glutamate analogue) is significantly increased in the presence of GFAP and NHERF1. Moreover, we demonstrate in vivo that expression of GFAP is essential in retaining GLAST in the plasma membranes of astrocytes after an hypoxic insult. These data indicate that the cytoskeleton of the astrocyte plays an important role in protecting the brain against glutamate-mediated excitotoxicity.