BACKGROUND: Korean cactus Cheonnyuncho (Opuntia humifusa) is rich in pectin, phenols, flavonoids, and minerals such as calcium and phosphorus. Some Koreans drink Cheonnyuncho juice prepared by grinding Cheonnyuncho with water. Cheonnyuncho is well known for its functional properties and antioxidant effects, but its effect on constipation has not been sufficiently studied. METHODS: Loperamide (2 mg/kg) was injected subcutaneously to induce constipation in rats. The animals were divided into four groups: a normal group (NOR), constipation control group (CON), and two constipation groups receiving the Cheonnyuncho extract (CE) at two different concentrations in drinking water, 3% (L-CE group) and 6% (H-CE group), for 25 days. RESULTS: The fecal pellet numbers of NOR and L-CE were significantly increased from 35.67 ± 2.09 (CON) to 50.60 ± 1.38 and 46.50 ± 2.91 after loperamide treatment, respectively (p < 0.05). The water content of fecal excretions was significantly enhanced in only the L-CE group (33.05 ± 0.49%) compared to control (23.38 ± 1.26%) (p < 0.05) after loperamide treatment. The oral intake of CE (L-CE and H-CE groups) significantly increased levels of the intestinal transit ratio (45.25 ± 1.86% and 41.05 ± 2.47%, respectively) compared to the CON group (32.15 ± 2.05%) (p < 0.05). Treatment with the low concentration of CE significantly increased fecal levels of acetic, propionic, butyric, and valeric acids, as well as the total short-chain fatty acid (SCFA) concentration. Histological analyses revealed that the thickness of the distal colon also increased in the CE-treated groups in a dose-dependent manner. CONCLUSIONS: Constipation decreased when CE was fed to the rats. In particular, the fecal pellet number and water content, as well as histological parameters such as distal colon thickness, improved. The CE treatment also increased the fecal SCFA content. These results show that the extract of Cheonnyuncho (O. humifusa) alleviated the symptoms of loperamide-induced constipation.