Interactions between microbiota and gastrointestinal (GI) function may be evident from the early stage of life. Microbiota contributes to myenteric nerve development and normal gut function maintenance. Earlier studies suggested that germ-free or antibiotics treated mice have decreased gastrointestinal transit. Also, abnormal gut microbiota is common in patients with altered GI motility, like IBS. However, the underlying mechanism are not fully understood. Here, we investigated the role of microbiota in GI motility in vivo and vitro. Specific-pathogen-free (SPF) mice and germ-free (GF) mice (12–16 weeks old) were used. Gastric emptying was assessed by videofluoroscopy and GI transit was assessed by SHAPE study using metallic beads, as described previously. Mice were then sacrificed and samples from jejunum, ileum and mid-colon were taken. Isometric contraction was assessed after electric field stimulation (EFS) and pharmacological treatments. Acetylcholine (Ach) release was assessed by superfusion using isotope [3H]. Expression of genes related to neuromuscular function was assessed by Illumina, immunostaining was also performed. There were no significant difference in gastric emptying rates between SPF and GF mice, which were 26.4±4.1 % (n=10) and 23.1±3.1 % (n=11), respectively. However, GI transit (total scores) was faster in SPF mice compared to GF mice, with 13.6±1.7 (n=10) and 5.4±0.2 (n=11) respectively. Beads were mainly located in the cecum in SPF mice, but in GF mice, most of them remained in the proximal small intestine. In contractility studies, carbachol induced similar level of contraction in SPF or GF tissues, both in the small bowel and colon. In SPF tissues, the neural stimulation caused a transient contraction in jejunum, and biphasic relaxation and contraction in ileum and colon. In GF tissues, neural stimulation caused similar response in ileum and colon, however, in jejunum, it caused biphasic response with a significant relaxation followed by a contraction. No difference was seen in EFS-induced Ach release between SPF and GF tissues. Microbiota appears to be essential in the establishment of normal small intestinal motility. Germ-free mice have altered jejunal motility, likely due to a non-cholinergic neural response. Gene expression study and immunostaining are underway to reveal the underlying mechanism. None