As a traditional Chinese herbal medicine, Anemarrhena asphodeloides Bge. possesses the effects of nourishing yin, moistening dryness, clearing lungs and relieving fire. Simultaneously, it has been used to treat constipation for more than one thousand years in China. However, modern medical studies are limited and lacking on its therapeutic mechanism. Aim of the study: This current study was aimed to investigate the laxative activities and explore the potential mechanism of Anemarrhena asphodeloides Bge. polysaccharides (AABP) in loperamide-induced constipation rats. The structure of AABP was determined by using infrared spectrum, high performance gel permeation chromatography (HPGPC), and high performance liquid chromatography (HPLC). Real-time quantitative polymerase chain reaction (PCR), multitudinous methods were adopted to explore the underlining therapeutic mechanism of AABP in treating constipation, including enzyme-linked immunosorbent assay (ELISA), histopathological, immunohistochemistry and western blotting. In the present study, the average molecular weight of AABP was determined as 1.11 × 103 kDa. The primary monosaccharide compositions were analyzed including D-mannose, L-rhamnose, D-galacturonic acid, D-glucose, D-galactose and L-arabinose (1, 0.04, 0.53, 0.11, 0.33, 0.25, respectively) by high-performance liquid chromatography (HPLC). AABP significantly increased the levels of gastrin (Gas), motilin (MTL), substance P (SP), 5-hydroxytryptamine (5-HT) and vasoactive intestinal peptide (VIP), and decreased the NO content of loperamide-induced rats to ameliorate constipation in the rats. Whilst, AABP repaired the damaged colons by regulating PCNA and ICAM-1 protein expressions. Additionally, AABP up-regulated the levels of SCF, c-Kit, AQP3 and VIP as well as down-regulated the expressions of AQP8, AQP4 and PGE2. The present findings suggested that AABP were the laxative active ingredients isolated from Anemarrhena asphodeloides Bge., which could treat constipation through regulating the gastrointestinal hormones and neurotransmitters to improve the intestinal motility and water metabolism. Display omitted