Much remains unknown about the role of gut microbes in the development of a functional enteric nervous system (ENS), a network of neurons and glia within the gastrointestinal tract (GI) that is fundamental to motility. We hypothesized that that many cases of chronic dysmotility in adults result in part from disturbances to gut microbes during a critical window of development—more generally, that microbial stimuli influence ENS development and thus long-term GI motility. In this study, microbe-lacking (“germ-free”, GF) mice, which are known to have extremely slow GI transit, were given fecal microbiota transplantation (FMT) at weaning or as adults. We found that only the mice given FMT at weaning appeared to achieve normal overall transit, while those given FMT as adults failed to regain normal transit, showing only limited improvements. Providing clues into the mechanistic underpinnings of these functional differences, RNAseq of colonic muscularis propria revealed enrichments in neuron developmental pathways in mice exposed to gut microbes earlier in life, while mice exposed later – or not at all – showed exaggerated expression of inflammatory immune pathways. These findings highlight a microbiota-dependent sensitive period in ENS development, pointing to potential roles of the early life microbiome in later life dysmotility.