Significance: The gut microbiota affects several physiological processes, including gut motility. Here we observed that germ-free mice have an immature enteric nervous system (ENS) that is normalized upon colonization with a normal microbiota. We identified the mechanism of communication between the microbiota and enteric neurons as the initiation of serotonin release and subsequent activation of the 5-HT ₄ receptor. This demonstrates a strong interaction between the microbiota and the ENS and indicates potential mechanisms linking microbial dysbiosis to gastrointestinal disorders. The ability to modulate the microbiota, e.g., by diet, will open new perspectives of research in neurogastroenterology.