Enteric glia (EG) in the enteric nervous system can modulate neuronally regulated gut functions. Using molecular genetics, we assessed the effects that molecular entities expressed in EG and otherwise mediating two distinct mechanisms of gliotransmitter release, connexin 43 (Cx43) hemichannel vs. Ca2+-dependent exocytosis, have on gut function. The expression of mutated Cx43(G138R) (which favors hemichannel, as opposed to gap-junctional activity) in EG increased gut motility in vivo, while a knock-down of Cx43 in EG resulted in the reduction of gut motility. However, inhibition of Ca2+-dependent exocytosis in EG did not affect gut motility in vivo; rather, it increased the fecal pellet fluid content. Hampering either Cx43 expression or Ca2+-dependent exocytosis in EG had an effect on colonic migrating motor complexes, mainly decreasing frequency and velocity of contractions ex vivo. Thus, EG can differentially modulate gut reflexes using the above two distinct mechanisms of gliotransmission.