PURPOSE: To investigate the therapeutic effects of protease-activated receptor 2 (PAR-2) agonist SLIGRL-NH on loperamide-induced Sprague-Dawley (SD) rat constipation animal models. MATERIALS AND METHODS: Loperamide was injected subcutaneously to induce constipation twice a day for 3 days. SD rats (n = 30) were randomly divided into five groups: non-constipation group (control, n = 6), constipation group (constipation, n = 6), constipation + SLIGRL-NH low-dosage group (SLIGRL-NH low, n=6), constipation + SLIGRL-NH high-dosage group (SLIGRL-NH high, n = 6), and constipation + prucalopride (positive control, n = 6). The SLIGRL-NH low group and SLIGRL-NH high group were administered with 2.5 μmol/kg and 5 μmol/kg SLIGRL-NH, respectively, and the prucalopride group received 2 mg/kg prucalopride. The control and constipation group received 1× PBS under the same pattern. SLIGRL-NH and prucalopride were orally administrated once daily for 7 days. On the final day of oral administration, food intake, water intake, the number of stool pellets, weight, and fecal water content was calculated; moreover, the colons of rats in different groups were collected and histological features were examined by hematoxylin and eosin staining; furthermore, the expression of anoctamin-1 was determined by Immunohistochemical methods, and the expressions of c-kit and PAR-2 were examined using real-time quantitative polymerase chain reaction and Western blot methods; finally, the expressions of neurotransmitter vasoactive intestinal peptide (VIP) and substance P (SP) were examined using enzyme-linked immuno-sorbent assay methods. RESULTS: The feeding and excretion behaviors, intestinal transit ratio, and the histological feature of the colon in the constipated rats were all improved by SLIGRL-NH treatment; moreover, SLIGRL-NH treatment induced significant increase in the expression of PAR-2 and also increased number of interstitial Cajal cells. Furthermore, SLIGRL-NH also decreased the contents of the inhibitory neurotransmitter VIP and increased the expression of the excitatory neurotransmitter SP. High dose of SLIGRL-NH has shown similar anti-constipation effects as prucalopride. CONCLUSION: These results suggested that SLIGRL-NH can enhance gastrointestinal transit and alleviate in rats with loperamide-induced constipation.