Background & Aims In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca2+ responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca2+-mediated responses in enteric glia are required to maintain gastrointestinal function. Methods We used in situ Ca2+ imaging to monitor glial Ca2+ responses, which were manipulated with pharmacologic agents or via glia-specific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreERT2+/-/Cx43f/f mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses. Results Ca2+ responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca2+ responses. In vivo attenuation of Ca2+ responses in the enteric glial network slowed gut transit overall and delayed colonic transit - these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca2+-mediated responses and changes in glial expression of Cx43 messenger RNA and protein. Conclusions Ca2+-mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders. © 2014 by the AGA Institute.