BACKGROUND AND AIMS: The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R), triggering ATP release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca(2+)]i) responses in the surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation. METHODS: We studied neuroinflammation in vivo using the 2,4-dinitrobenzenesulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP∷Cre (ERT2+/-)/Cx43(f/f) ] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOS (-/-)) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca(2+) and NO imaging were used to monitor glial [Ca(2+)]i and [NO]i. RESULTS: Purinergic activation of enteric glia drove [Ca(2+)]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO. CONCLUSIONS: Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders.