Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on non-neuronal cells of the CNS. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17-estradiol can activate rapid signaling and modulate non-classical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with hER (C6ER) or rER (C6ER). Introduction of a CRE-luc reporter gene into C6, C6ER and C6ER cells, lead to the observation that 17-estradiol treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ER, but had no effect on reporter gene expression in C6ER or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA) suggesting the modulation of non-classical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (PI3K) or U73122 (PLC) attenuated the 17-estradiol-induced reduction in non-classical transcription. We conclude that 17-estradiol treatment reduces CRE-mediated transcription in glioma cells expressing ER and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of non-classical transcription.