ABSTRACT Abdominal pain is the most common gastrointestinal issue and is a major cause of suffering in functional gastrointestinal disorder such as irritable bowel syndrome (IBS) and in the inflammatory bowel diseases (IBD). Despite extensive efforts, there is still no consensus regarding the mechanisms responsible for producing abdominal pain. This lack of progress is reflected by the poor efficacy of current therapies and the stalled progress toward novel, targeted therapies for abdominal pain. The overall goal of this proposal is to understand mechanisms that sensitize visceral nerve fibers and the specific focus of this proposal is on mechanisms regulated by enteric glia. Glia play a central role in the regulation of pain transmission in the central nervous system, but how interactions between glia and nerve fibers in the intestine contribute to the generation of visceral pain is unknown. This proposal tests the central hypothesis that enteric glia contribute to visceral hypersensitivity by direct interactions with nociceptors, and indirectly by modulating immune responses. This dual hypothesis will be tested in two specific aims that utilize targeted genetic models to manipulate glia, established animal models of visceral hypersensitivity, and optogenetic recordings, immunohistochemical assays, electrophysiology, and visceromotor reflex recordings to study the impact on sensory neuron activity in health and disease. Aim 1 will test the hypothesis that enteric glia directly modulate the activity of visceral nociceptors. Specific Aim 1a will use chemogenetic and knockout mouse models to study the role of gliotransmitter release and Specific Aim 1b will use selective drugs and knockout mice to study the role of glial ectoenzymes that regulate ATP and histamine availability. Aim 2 will test the hypothesis that enteric glia indirectly modulate visceral nociceptors through interactions with immune cells. Specific Aim 2a will use transgenic mice and monoclonal antibodies to study glial interactions with macrophages mediated by the release of mediators including ATP and M-CSF. Specific Aim 2b will use knockout mice and monoclonal antibodies to study interactions that are secondarily dependent on interactions between glia and lymphocytes mediated by antigen presentation. Immune responses in Aim 2 will be analyzed by microarrays, immunohistochemistry, and flow cytometry. The results of this study will identify novel effects of glial–immune interactions on sensory neurons in the periphery. This new insight into mechanisms that sensitize visceral nerves will facilitate the development of new therapies for abdominal pain in functional gastrointestinal disorders, such as IBS and IBD.