Constipation is a common gastrointestinal disorder often treated symptomatically as the existing therapies all have significant side-effects. The relaxin/insulin-like family peptide receptor 4 (RXFP4), a class A G protein-coupled receptor, has emerged as a potential drug target for this indication as it regulates intestinal motility. Here, we investigated the therapeutic effects of both peptidic (insulin-like peptide 5, INSL5) and small molecule (DC591053) RXFP4 agonists on loperamide-induced constipation in mice. Fecal water content, fecal weight, colonic transit time, and serum levels of neurotransmitters such as nitric oxide (NO), serotonin (5-HT), and vasoactive intestinal peptide (VIP) were assessed, in conjunction with the examination of colon tissue histology and expression levels of aquaporin 3 (AQP3), transient receptor potential vanilloid 1 (TRPV1), and calcitonin gene-related peptide (CGRP). It was found that both INSL5 and DC591053 dose-dependently increased fecal water content and weight, accelerated colonic transit, and improved colon morphology in constipated mice, accompanied by the altered expression levels of factors related to constipation. Comparative analysis revealed that while the two agonists produced similar beneficial outcomes, their different chemical nature may affect pharmacokinetics property and receptor engagement. Our results suggest that the activation of RXFP4 presents a novel approach to alleviating constipation.