Key points Cholera causes more than 100,000 deaths each year as a result of severe diarrhoea, vomiting and dehydration due to the actions of cholera toxin; more females than males are affected. Cholera toxin induces hypersecretion via release of mucosal serotonin and over‐activation of enteric neurons, but its effects on gastrointestinal motility are not well characterized. We found that cholera toxin rapidly and reversibly reduces colonic motility in female mice in oestrus, but not in males or females in prooestrus, an effect mediated by 5‐HT in the colonic mucosa and by 5‐HT3 receptors. We show that the number of mucosal enterochromaffin cells containing 5‐HT changes with the oestrous cycle in mice. These findings indicate that cholera toxin's effects on motility are rapid and depend on the oestrous cycle and therefore can help us better understand differences in responses in males and female patients. Abstract Extensive studies of the mechanisms responsible for the hypersecretion produced by cholera toxin (CT) have shown that this toxin produces a massive over‐activation of enteric neural secretomotor circuits. The effects of CT on gastrointestinal motility, however, have not been adequately characterized. We investigated effects of luminal CT on neurally mediated motor activity in ex vivo male and female mouse full length colon preparations. We used video recording and spatiotemporal maps of contractile activity to quantify colonic migrating motor complexes (CMMCs) and resting colonic diameter. We compared effects of CT in female colon from wild‐type and mice lacking tryptophan hydroxylase (TPH1KO). We also compared CMMCs in colons of female mice in oestrus with those in prooestrus. In female (but not male) colon, CT rapidly, reversibly and concentration‐dependently inhibits CMMC frequency and induces a tonic constriction. These effects were blocked by granisetron (5‐HT3 antagonist) and were absent from TPH1KO females. CT effects were prominent at oestrus but absent at prooestrus. The number of EC cells containing immunohistochemically demonstrable serotonin (5‐HT) was 30% greater in female mice during oestrus than during prooestrus or in males. We conclude that CT inhibits CMMCs via release of mucosal 5‐HT, which activates an inhibitory pathway involving 5‐HT3 receptors. This effect is sex‐ and oestrous cycle‐dependent and is probably due to an oestrous cycle‐dependent change in the number of 5‐HT‐containing EC cells in the colonic mucosa.