Glutamate is a critical neurotransmitter of the central nervous system (CNS) and also an important regulator of cell survival and proliferation. The binding of glutamate to metabotropic glutamate receptors induces signal transduction cascades that lead to gene-specific transcription. The transcription factor NF-B, which regulates cell proliferation and survival, is activated by glutamate; however, the glutamate receptor-induced signaling pathways that lead to this activation are not clearly defined. Here we investigate the glutamate-induced activation of NF-B in glial cells of the CNS, including primary astrocytes. We show that glutamate induces phosphorylation, nuclear accumulation, DNA binding, and transcriptional activation function of glial p65. The glutamate-induced activation of NF-B requires calcium-dependent IB kinase (IKK) and IKKβ activation and induces p65-IB dissociation in the absence of IB phosphorylation or degradation. Moreover, glutamate-induced IKK preferentially targets the phosphorylation of p65 but not IB. Finally, we show that the ability of glutamate to activate NF-B requires cross-coupled signaling with the epidermal growth factor receptor. Our results provide insight into a glutamate-induced regulatory pathway distinct from that described for cytokine-induced NF-B activation and have important implications with regard to both normal glial cell physiology and pathogenesis.