We report for the first time the distribution of human INSL5 and its cognate leucine rich G-protein coupled receptor RXFP4 in the large intestine and in neuroendocrine/carcinoid tissues. Immunoreactive INSL5 was uniquely expressed by enteroendocrine cells (EECs) located within the colonic mucosa, whereas colonocytes were immunopositive for RXFP4. INSL5+ and RXFP4+ cells were also detected in human neuroendocrine/carcinoid tissues. We employed a recently described Insl5 knockout mouse model and 2 mouse models of induced colitis to address the relevance of Insl5 in EEC development and in acute inflammation of the colon. We identified INSL5 as a specific marker for synaptophysin+ EECs in the mucosa of the normal human and mouse colon. Insl5 was not essential for the development of mouse synaptophysin+ EECs. The mouse models of chemically induced colitis (dextran sulfate sodium and dinitrobenzene-sulfonic acid) failed to show changes in the numbers of Insl5+ EECs at inflammatory sites during the acute phase of colitis. In conclusion, we showed that INSL5 is a novel marker of colorectal EECs and provide first evidence for the presence of a potentially autocrine/paracrine INSL5-RXFP4 signaling system in the normal human and mouse colon and in rare human neuroendocrine tumours.