•  Previous studies have indicated that neither neuronal nor mucosal 5‐hydroxytryptamine (5‐HT) are important for colonic migrating motor complexes (CMMCs) or faecal pellet propulsion. Therefore, tryptophan hydroxylase 1 knockout (TPH1KO) mice were used to examine the role of mucosal 5‐HT in generating CMMCs and faecal pellet propulsion, as TPH1 is the regulatory enzyme necessary for the synthesis of 5‐HT in enterochromaffin cells in the mucosa. •  Control mice generated a robust CMMC when the mucosa was mechanically stimulated, which was blocked by ondansetron (5‐HT3 antagonist), and could propagate faecal pellets that did not significantly distend the bowel, suggesting that they were propelled by mucosal reflexes in the absence of stretch reflexes. •  TPH1KO mice exhibited no mucosal reflexes, reduced responses to intraluminal distension and propelled only larger faecal pellets, suggesting that they relied upon stretch reflexes alone. •  In control mice, CMMCs, which can propel a faecal pellet, propagated in an oral to anal direction, whereas, in TPH1KO mice, they rarely propagated. •  Both the propagation and amplitude of CMMCs were reduced by ondansetron in control mice, whereas this drug did not affect CMMCs in TPH1KO mice. •  This suggests that 5‐HT release from the mucosa and stretch reflexes are important for normal colonic propulsion. Abstract  Although there is general agreement that mucosal 5‐hydroxytryptamine (5‐HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5‐HT is critical. We therefore tested the hypothesis that the secretion of 5‐HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1−/− mice), which is the rate‐limiting enzyme in the biosynthesis of mucosal but not neuronal 5‐HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1+/+) mice, Tph1−/− animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1+/+ colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1+/+); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1−/− colon; and (9) the 5‐HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1+/+ mice, failed to alter CMMCs in Tph1−/− animals. Our observations suggest that mucosal 5‐HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon.