Insulin-like peptide 5 (INSL5) is a gut hormone that is exclusively produced by colonic L-cells. We have recently developed analogues of INSL5 that act as agonists for the relaxin family peptide receptor 4 (RXFP4) both in vitro and in vivo. We showed that INSL5-A13, an RXFP4 agonist, stimulates colorectal propulsion in wild-type mice, but not in RXFP4 knockout mice. These results suggest that INSL5 may have a physiological role in the control of colorectal motility. To investigate this possibility, we have recently designed and developed a novel INSL5 analogue, INSL5-A13NR. This compound is a potent antagonist in two in vitro assays and blocks agonist-induced increase in colon motility in mice that express RXFP4. Our data also show that colorectal propulsion induced by intracolonic administration of bacterial products (short-chain fatty acids, SCFAs) is antagonized by INSL5-A13NR. Our studies, therefore, suggest that RXFP4 is a potential drug target for the treatment of colon motility disorders such as constipation and diarrhea.