Obesity alters gastrointestinal (GI) motility and 5‐HT signalling. Altered 5‐HT signalling disrupts control of GI motility. Levels of extracellular 5‐HT depend on enterochromaffin (EC) cell release and serotonin transporter (SERT) uptake. We assessed GI transit and 5‐HT signalling in the jejunum of normal and obese mice. Male and female mice were fed a control diet (CD; 10% of kilocalories as fat) or a high‐fat diet (HFD; 60% of kilocalories as fat). Gastrointestinal transit was increased in male HFD‐fed and female CD‐fed compared with male CD‐fed mice. The 5‐HT₃ receptor blocker, alosetron, increased gastric emptying in male CD‐fed mice, but decreased transit in female CD‐fed mice. The 5‐HT‐induced jejunal longitudinal muscle contractions in vitro were similar in all mice. In contrast to male CD‐fed mice, 5‐HT uptake (measured using continuous amperometry in vitro) in male HFD‐fed mice was fluoxetine insensitive, yet sensitive to cocaine and the dopamine transporter (DAT) blocker GBR 12909. Immunoreactivity for DAT was present in the mucosa, and protein levels were greater in male HFD‐fed compared with CD‐fed mice. Extracellular 5‐HT and mucosal 5‐hydroxyindolacetic acid (5‐HT metabolite) were similar in male HFD‐fed compared with CD‐fed mice. 5‐Hydroxytryptamine uptake was fluoxetine sensitive in all females. Greater SERT protein, decreased extracellular 5‐HT and greater mucosal 5‐hydroxyindolacetic acid were observed in female HFD‐fed compared with CD‐fed mice. Mucosal 5‐HT and EC cell numbers were similar in CD‐fed and HFD‐fed mice of both sexes; female 5‐HT and EC cell numbers were increased compared with males. The HFD did not alter plasma sex hormone levels in any mice. Overall, obesity alters GI transit and 5‐HT signalling in a sex‐dependent manner.