Inflammatory bowel disease (IBD) is characterized by pain, bleeding, cramping and altered gastrointestinal (GI) function. Changes in mucosal 5‐HT (serotonin) signalling occur in animal models of colitis and in humans suffering from IBD. Melatonin is co‐released with 5‐HT from the mucosa and has a wide variety of actions in the GI tract. Here, we examined how melatonin signalling is affected by colitis and determined how this relates to 5‐HT signalling. Experimental Approach Using electroanalytical approaches, we investigated how 5‐HT release, reuptake and availability as well as melatonin availability are altered in dextran sodium sulfate (DSS)‐induced colitis in mice. Studies were conducted to explore if melatonin treatment during active colitis could reduce the severity of colitis. Key Results We observed an increase in 5‐HT and a decrease in melatonin availability in DSS‐induced colitis. A significant reduction in 5‐HT reuptake was observed in DSS‐induced colitis animals. A reduction in the content of 5‐HT was observed, but no difference in tryptophan levels were observed. A reduction in deoxycholic acid‐stimulated 5‐HT availability and a significant reduction in mechanically‐stimulated 5‐HT and melatonin availability were observed in DSS‐induced colitis. Orally or rectally administered melatonin once colitis was established did not significantly suppress inflammation. Conclusion and Implications Our data suggest that DSS‐induced colitis results in a reduction in melatonin availability and an increase in 5‐HT availability, due to a reduction/loss of tryptophan hydroxylase 1 enzyme, 5‐HT content and 5‐HT transporters. Mechanosensory release was more susceptible to inflammation when compared with chemosensory release.