Serotonin (5‑hydroxytryptamine; 5‑HT) may be a key player in gastrointestinal (GI) motility and the GI immune system. In the present study, the effect of gut microbiota on the association between GI motility, and 5‑HT expression and macrophage abundance in the GI tract was examined. Germ‑free (GF) mice (6 weeks old) were orally administered a fecal bacterial suspension prepared from specific pathogen‑free mice and their GI tissues were evaluated 4 weeks later. The expression of 5‑HT and mannose receptor (MR) was examined by immunohistochemistry, and GI transit time (GITT) was measured by administration of carmine red solution. The numbers of 5‑HT‑positive endocrine cells and muscularis MR‑positive macrophages were significantly increased in the upper GI and colon of GF mice subjected to fecal transplantation (FT) compared with control GF mice without FT. GITT was significantly decreased in GF mice subjected to FT compared with GF mice without FT, and negatively correlated with the numbers of 5‑HT‑positive cells in the upper GI and muscularis MR‑positive macrophages throughout the GI tract. The numbers of 5‑HT‑positive endocrine cells and muscularis MR‑positive macrophages were significantly correlated throughout the GI tract. The present results suggest that the gut microbiota is involved in the association between accelerated GI motility and induction of the 5‑HT/muscularis MR‑positive macrophage axis in the GI tract.