Abstract: Objective: To investigate the laxative effects of aqueous extracts of the stems and leaves of Glehniae Radix on constipated mice. In addition, this study aimed to provide data to support the development and utilization of the stems and leaves of Glehniae Radix. Methods: A mouse model of constipation was established using loperamide (10 mg/kg/d) for 7 d, and the mice were treated with low and high doses of aqueous extracts from the stems and leaves of Glehniae Radix (4 and 8 g/kg/d) administered through gavage for 7 d. Metabolic indices of mice were determined, and morphological changes in colon tissues were observed using hematoxylin-eosin (H&E) staining. Alcian Blue (AB) and Alcian Blue/periodic acid-chiff (AB-PAS) staining were used to evaluate goblet cells and mucus secretion in the colon tissues. Serum nitric oxide (NO) and 5-hydroxytryptamine (5-HT) concentrations were measured using kits. Immunohistochemical (IHC) analysis was performed to assess the expression of vasoactive intestinal peptide (VIP) and aquaporin 3 (AQP3) in colon tissues. Results: Compared to model group, the time to the first white stool excretion in the high-dose group was significantly reduced (P<0.001), fecal water content after 6 h was significantly elevated (P<0.05), and serum 5-HT levels were significantly increased (P<0.01). Compared with the model group, mice in both the low- and high-dose groups exhibited a significant increase in the small intestinal propulsion rate (P<0.001) and colonic tissue pathology was alleviated. Mucin secretion in the colon was elevated and colon goblet cell was significantly increased (P<0.001) and serum NO levels were significantly reduced (P<0.001). In addition, VIP expression was significantly increased (P<0.05, P<0.01), whereas AQP3 expression was significantly decreased (P<0.05, P<0.01) in the colonic tissues. Conclusion: The aqueous extract of Glehniae Radix stems and leaves markedly enhanced intestinal motility in constipated mice, mitigated colonic pathological damage, and restored the mucosal barrier, thereby exerting a laxative effect. Its mechanism may involve the regulation of intestinal mucus secretion and water transport, improving intestinal motility by increasing serum 5-HT levels and colonic VIP expression, and decreasing serum NO levels and AQP3 expression, thereby alleviating constipation.