Abstract: Objective: To study the effect of mulberry leaf water extract on isolated ileum contractile activity of guinea pig and constipation rats, and to explore the mulberry leaf improving constipation effects and related mechanisms. Methods: The effect of mulberry leaf water extract on isolated ileum movement of guinea pigs was observed by measuring in vitro intestinal muscle tension. Loperamide (10 mg/kg/d) was used to construct 3 d and 7 d constipation rat models, and mulberry leaf water extract (5 g/kg/d) was administered by gavage at the same time to observe the time of first defecation, fecal number for 24 h and small intestine propulsion rate of rats. The ileum contractile reaction induced by carbachol was measured. The histological changes of colon were observed by HE staining of paraffin section, and the expression of AQP3 in colon was detected by immunohistochemistry. Results: Mulberry leaf water extract promoted the voluntary movement of ileum in guinea pigs, which was manifested by increased contraction amplitude and mean tension. The first defecation time, fecal number at 24 h and small intestine propulsion rate were improved in both group of mulberry leaf 3 d and 7 d. Mulberry leaf water extract significantly improved the reduction of the average tension of the isolated ileum induced by carbachol in constipation rats. Mulberry leaf water extract improved the musculature thinning of colon tissue caused by loperamide, and significantly reduced the increase in colon AQP3 expression casued by loperamide. Conclusion: Mulberry leaf water extract can significantly promote ileum movement in guinea pigs, and improve constipation in rats, which may through reducing the expression of AQP3, inhibiting the excessive absorption of colonic water, increasing the water content of feces, and promoting intestinal muscle movement.