Background: The 5-hydroxytrptamine receptor 4 (5-HT4 receptor) is heavily expressed on colonic epithelial cells and has been targeted as a therapeutic for functional bowel symptoms and pain; however, adverse cardiac events related to 5-HT4 agonist treatment limited their therapeutic use. Previous studies in the Mawe laboratory have demonstrated that intraluminal application of a 5-HT4 agonist exerts protective epithelial actions in animal models of colitis, and accelerates recovery from colitis. The aim of this study was to test the effects of a luminally restricted 5-HT4 agonist in a mouse model of experimental colitis. Methods: The luminally restricted 5-HT4 agonist (Takeda Pharmaceuticals; 10 mg/kg) was administered to mice during active dextran sodium sulfate (DSS) induced colitis. Colitis activity was evaluated using disease activity index, a fecal lipocalin-2 assay, and histological damage scoring. Epithelial proliferation and colonic motility were also measured as readouts of the potential protective actions and colonic function, respectively. Results: Oral gavage and intracolonic delivery of this luminally restricted 5-HT4 agonist had no detectable effect on recovery from colitis or colonic motility as compared to vehicle. Additionally, in positive control experiments, we failed to see an effect of the 5-HT4 agonist, tegaserod, on colitis severity or colonic motility in any of the measures tested. Conclusions: In conclusion, it is unclear if the luminally restricted 5-HT4 agonist has any effect on recovery from DSS colitis. Given inconsistencies with the model and lack of an effect of tegaserod, additional studies will be required, possibly involving different doses and time points, to fully assess the actions of this luminally restricted compound in colitis recovery.