Flammulina velutipes, (Curt. ex Fr.) Sing, a popularly edible fungus, has been widely used both as a restorative drug and a tonic food in China. In the current study, the effect of F. velutipes polysaccharides was evaluated with a constipated rat model induced by loperamide hydrochloride. The rats were divided into six groups: normal group, model group, positive control group, F. velutipes polysaccharides high, moderate and low dose groups. Rats were given 2 mL of Loperamide hydrochloride (3 mg·kg·d), by intragastric administration 2 times per day for 10 days. F. velutipes polysaccharides at the doses of 600, 400 and 200 mg/kg (1 mL/100g weight) were administered to treat rats with constipation for 7 days. The gastrointestinal hormones, including motilin (MTL), gastrin (GAS), substance P (SP), somatostatin (SS), and intestinal propulsive rate and feces weight at 24 hours after treatment were used as the indexes to evaluate the effects of F. velutipes polysaccharides on constipation. The levels of MTL, GAS and SP in serum significantly increased and the levels of SS in serum of rats significantly decreased after the treatment of rats with F. velutipes polysaccharides as compared with those of rats in the model group.