Short chain fatty acids (SCFAs) accelerate colonic transit. This study examined whether this action was mediated by activation of the peristaltic reflex. SCFAs (acetate, butyrate, or propionate) were applied to the central compartment of a three-compartment flat-sheet preparation of rat mid-distal colon. Release of 5-HT, BDNF, and CGRP was measured in all three compartments. Ascending contraction and descending relaxation were measured in the orad and caudad compartments. Addition of SCFAs at physiological to supraphysiological concentrations (0.5 to 100 mM) to the central compartment elicited concentration-dependent ascending contraction and descending relaxation (EC50 ~ 5 mM). At this concentration, the SCFAs induced 8- to 11-fold increase in 5-HT release and 2- to 3-fold increase in CGRP release in the central compartment only. They had no effect on BDNF release. CGRP release was inhibited by a 5-HT₄ but not a 5-HT₃ receptor antagonist. Ascending contraction and descending relaxation were also inhibited by 5-HT₄ and by CGRP receptor antagonists to the central compartment. 5-HT and CGRP release, as well as ascending contraction and descending relaxation induced by mechanical stimulation of the mucosa (2 to 8 strokes) were significantly augmented by 1 mM acetate. Acetate (1 mM) also doubled propulsive velocity in isolated whole segments of guinea pig colon. In conclusion, chemical stimulation of the mucosa by SCFAs triggers a peristaltic reflex mediated by release of 5-HT from mucosal cells and activation of 5-HT₄ receptors on sensory CGRP-containing nerve terminals. This SCFA-induced peristaltic pathway augments the peristaltic reflex elicited by mechanical stimulation of the mucosa.